Permalink
Cannot retrieve contributors at this time
Fetching contributors…
Cannot retrieve contributors at this time
| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">01340</article-id><article-id pub-id-type="doi">10.7554/eLife.01340</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Biochemistry</subject></subj-group><subj-group subj-group-type="heading"><subject>Biophysics and structural biology</subject></subj-group></article-categories><title-group><article-title>Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-6889"><name><surname>Nachtergaele</surname><given-names>Sigrid</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-8"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-6992"><name><surname>Whalen</surname><given-names>Daniel M</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-9"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" id="author-6993"><name><surname>Mydock</surname><given-names>Laurel K</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" id="author-6994"><name><surname>Zhao</surname><given-names>Zhonghua</given-names></name><xref ref-type="aff" rid="aff4"/><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" id="author-6995"><name><surname>Malinauskas</surname><given-names>Tomas</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-9"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" id="author-6996"><name><surname>Krishnan</surname><given-names>Kathiresan</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/><xref ref-type="other" rid="dataro2"/></contrib><contrib contrib-type="author" id="author-6997"><name><surname>Ingham</surname><given-names>Philip W</given-names></name><xref ref-type="aff" rid="aff4"/><xref ref-type="aff" rid="aff5"/><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-6998"><name><surname>Covey</surname><given-names>Douglas F</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-6999"><name><surname>Siebold</surname><given-names>Christian</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-5"/><xref ref-type="other" rid="par-6"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-6891"><name><surname>Rohatgi</surname><given-names>Rajat</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff6"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Biochemistry</institution>, <institution>Stanford University School of Medicine</institution>, <addr-line><named-content content-type="city">Stanford</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Division of Structural Biology</institution>, <institution>Wellcome Trust Centre for Human Genetics, University of Oxford</institution>, <addr-line><named-content content-type="city">Oxford</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff3"><institution content-type="dept">Department of Developmental Biology</institution>, <institution>Washington University School of Medicine</institution>, <addr-line><named-content content-type="city">St. Louis</named-content></addr-line>, <country>United States</country></aff><aff id="aff4"><institution>A*STAR Institute of Molecular and Cell Biology</institution>, <addr-line><named-content content-type="city">Singapore</named-content></addr-line>, <country>Singapore</country></aff><aff id="aff5"><institution>Lee Kong Chian School of Medicine, Imperial College London/Nanyang Technological University</institution>, <addr-line><named-content content-type="city">Singapore</named-content></addr-line>, <country>Singapore</country></aff><aff id="aff6"><institution content-type="dept">Department of Medicine</institution>, <institution>Stanford University School of Medicine</institution>, <addr-line><named-content content-type="city">Stanford</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Kuriyan</surname><given-names>John</given-names></name><role>Reviewing editor</role><aff><institution>Howard Hughes Medical Institute, University of California, Berkeley</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>christian@strubi.ox.ac.uk</email> (CS);</corresp><corresp id="cor2"><label>*</label>For correspondence: <email>rrohatgi@stanford.edu</email> (RR)</corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>29</day><month>10</month><year>2013</year></pub-date><pub-date pub-type="collection"><year>2013</year></pub-date><volume>2</volume><elocation-id>e01340</elocation-id><history><date date-type="received"><day>06</day><month>08</month><year>2013</year></date><date date-type="accepted"><day>26</day><month>09</month><year>2013</year></date></history><permissions><copyright-statement>© 2013, Nachtergaele et al</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Nachtergaele et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife01340.pdf"/><related-article ext-link-type="doi" id="ra1" related-article-type="commentary" xlink:href="10.7554/eLife.01680"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01340.001</object-id><p>The Hedgehog (Hh) signal is transduced across the membrane by the heptahelical protein Smoothened (Smo), a developmental regulator, oncoprotein and drug target in oncology. We present the 2.3 Å crystal structure of the extracellular cysteine rich domain (CRD) of vertebrate Smo and show that it binds to oxysterols, endogenous lipids that activate Hh signaling. The oxysterol-binding groove in the Smo CRD is analogous to that used by Frizzled 8 to bind to the palmitoleyl group of Wnt ligands and to similar pockets used by other Frizzled-like CRDs to bind hydrophobic ligands. The CRD is required for signaling in response to native Hh ligands, showing that it is an important regulatory module for Smo activation. Indeed, targeting of the Smo CRD by oxysterol-inspired small molecules can block signaling by all known classes of Hh activators and by clinically relevant Smo mutants.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.001">http://dx.doi.org/10.7554/eLife.01340.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01340.002</object-id><title>eLife digest</title><p>Just over 30 years ago, researchers identified a new signaling molecule with an important role in the development of fruit flies. Embryos lacking this molecule were thought to resemble a hedgehog, eventually leading to this cell–cell communication system being designated the “Hedgehog” pathway. This pathway has subsequently been shown to be involved in the development of many other animals, as well as in the repair of damaged tissues in adult organisms.</p><p>Abnormal Hedgehog signaling has also been implicated in both human birth defects and in cancers of the skin and the brain. Many such tumors are driven by the unrestrained activation of a membrane-bound protein called Smoothened, which has led to the development and clinical use of small molecules that prevent Hedgehog from activating Smoothened. The existing anti-tumor drugs all bind to the same region of the Smoothened receptor, namely the part that sits within the cell membrane. A second group of molecules, known as oxysterols, can activate Smoothened, but exactly how they do this has been unclear. Now, Nachtergaele et al. have shown that oxysterols bind to a region of the Smoothened receptor that lies outside the cell, and that is rich in the amino acid cysteine.</p><p>By solving the crystal structure of this part of the receptor from zebrafish, Nachtergaele et al. were able to map the oxysterol binding site at high resolution. This revealed strong similarities between this binding site and those in related receptors belonging to the Wnt signaling pathway. Deleting the cysteine-rich domain significantly impaired Hedgehog signaling, as did a new class of small molecule inhibitors designed specifically to target the oxysterol binding site.</p><p>In addition to providing new insights into the structure and function of the Smoothened receptor, the work of Nachtergaele et al. opens up possibilities for novel therapeutic agents that could be used in the treatment of cancers caused by abnormal Hedgehog signaling.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.002">http://dx.doi.org/10.7554/eLife.01340.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>Hedgehog signaling</kwd><kwd>oxysterol</kwd><kwd>smoothened</kwd><kwd>cysteine rich domain</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>Zebrafish</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>GM47969, HL67773</award-id><principal-award-recipient><name><surname>Krishnan</surname><given-names>Kathiresan</given-names></name><name><surname>Covey</surname><given-names>Douglas F</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>5 T32 HL007275</award-id><principal-award-recipient><name><surname>Mydock</surname><given-names>Laurel K</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Stand up to Cancer Foundation</institution></institution-wrap></funding-source><award-id>SU2C-AACR-IRG0209</award-id><principal-award-recipient><name><surname>Nachtergaele</surname><given-names>Sigrid</given-names></name><name><surname>Rohatgi</surname><given-names>Rajat</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>Pew Scholars in the Biomedical Sciences</institution></institution-wrap></funding-source><award-id>2009-000359-011</award-id><principal-award-recipient><name><surname>Nachtergaele</surname><given-names>Sigrid</given-names></name><name><surname>Rohatgi</surname><given-names>Rajat</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution>Wellcome Trust</institution></institution-wrap></funding-source><award-id>WT/082301/Z/07/Z</award-id><principal-award-recipient><name><surname>Siebold</surname><given-names>Christian</given-names></name></principal-award-recipient></award-group><award-group id="par-6"><funding-source><institution-wrap><institution>Cancer Research UK</institution></institution-wrap></funding-source><award-id>C20724/A14414</award-id><principal-award-recipient><name><surname>Siebold</surname><given-names>Christian</given-names></name></principal-award-recipient></award-group><award-group id="par-7"><funding-source><institution-wrap><institution>A*STAR Singapore</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Zhao</surname><given-names>Zhonghua</given-names></name><name><surname>Ingham</surname><given-names>Philip W</given-names></name></principal-award-recipient></award-group><award-group id="par-8"><funding-source><institution-wrap><institution>National Science Foundation</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Nachtergaele</surname><given-names>Sigrid</given-names></name></principal-award-recipient></award-group><award-group id="par-9"><funding-source><institution-wrap><institution>Medical Research Council</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Whalen</surname><given-names>Daniel M</given-names></name><name><surname>Malinauskas</surname><given-names>Tomas</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>A cysteine-rich domain within the Smoothened receptor may represent a novel therapeutic target for cancers caused by abnormal functioning of the Hedgehog signaling pathway.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>The Hedgehog (Hh) signaling pathway controls the development of many tissues during embryogenesis (<xref ref-type="bibr" rid="bib46">McMahon et al., 2003</xref>). Even quantitative abnormalities in Hh signaling can lead to human birth defects (<xref ref-type="bibr" rid="bib4">Bale, 2002</xref>). After development, Hh signaling regulates tissue stem cells and regenerative responses to injury (<xref ref-type="bibr" rid="bib41">Machold et al., 2003</xref>; <xref ref-type="bibr" rid="bib73">Shin et al., 2011</xref>). Aberrant Hh signaling can be oncogenic, and genes encoding Hh pathway proteins can function as oncogenes or tumor suppressor genes (<xref ref-type="bibr" rid="bib69">Scales and de Sauvage, 2009</xref>). The most commonly damaged step in Hh-driven cancers involves the poorly understood interaction between two transmembrane (TM) proteins, Patched 1 (Ptch1) and Smoothened (Smo) (reviewed in <xref ref-type="bibr" rid="bib9">Briscoe and Therond [2013]</xref>). Ptch1, encoded by a tumor suppressor gene, is a 12-pass TM protein that serves as the receptor for Hh ligands, including Sonic Hedgehog (Shh) (<xref ref-type="bibr" rid="bib42">Marigo et al., 1996</xref>; <xref ref-type="bibr" rid="bib75">Stone et al., 1996</xref>). In the absence of Hh ligands, Ptch1 inhibits the function of Smo, a 7-pass TM protein that is encoded by a human oncogene. Shh binds and inactivates Ptch1, unleashing Smo’s activity and allowing the Gli transcription factors to initiate target gene transcription. Despite the fact that Smo has become a drug target in oncology, with an FDA-approved Smo inhibitor in clinical use (<xref ref-type="bibr" rid="bib83">Von Hoff et al., 2009</xref>) and others in ongoing trials, the mechanism by which Smo is regulated by Ptch1 remains a mystery. Current models suggest that Ptch1, a protein with some homology to bacterial small molecule transporters, regulates Smo through an endogenous ligand whose identity is unknown (<xref ref-type="bibr" rid="bib19">Davies et al., 2000</xref>; <xref ref-type="bibr" rid="bib79">Taipale et al., 2002</xref>).</p><p>Smo consists of an extracellular N-terminal region containing a <underline>c</underline>ysteine <underline>r</underline>ich <underline>d</underline>omain (CRD), a heptahelical transmembrane segment (7TM) and an intracellular C-terminal tail (C-term) (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Smo belongs to the G-protein coupled receptor (GPCR) superfamily of proteins, most closely related to the Frizzled (Fz) group of Wnt receptors (<xref ref-type="bibr" rid="bib18">Dann et al., 2001</xref>; <xref ref-type="bibr" rid="bib30">Fredriksson et al., 2003</xref>). Previous work on Smo has largely focused on the 7TM domain, which contains a binding site for cyclopamine, a sterol-like plant alkaloid that was the foundational Hh inhibitor (<xref ref-type="bibr" rid="bib12">Chen et al., 2002a</xref>). A battery of subsequent small-molecule screens uncovered a set of exogenous ligands that regulate Smo activity through this site, either as agonists such as SAG or antagonists such as SANT-1 and the FDA-approved Hh-inhibitor Vismodegib (<xref ref-type="bibr" rid="bib29">Frank-Kamenetsky et al., 2002</xref>; <xref ref-type="bibr" rid="bib13">Chen et al., 2002b</xref>; <xref ref-type="bibr" rid="bib63">Robarge et al., 2009</xref>). The 2.5 Å crystal structure of the 7TM segment of Smo bound to a synthetic antagonist has provided a high-resolution view of this binding pocket, which is formed by the extracellular end of the 7TM helix bundle and connecting loops (<xref ref-type="bibr" rid="bib85">Wang et al., 2013</xref>). Smo drugs that occupy this ‘cyclopamine binding site’ are classified as such by their ability to compete with cyclopamine for Smo binding. No endogenous molecules are known that engage this site in the 7TM of Smo.<fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01340.003</object-id><label>Figure 1.</label><caption><title>The mouse Smo CRD is required to bind oxysterols.</title><p>(<bold>A</bold>) Schematic of full-length (FL), YFP-tagged mSmo and the ΔCRD and ΔC truncation mutants used in this study. (<bold>B</bold>) Structure of the 20(<italic>S</italic>)-OHC beads used in Smo pull-down assays. (<bold>C</bold>) EndoH and PGNaseF sensitivity of YFP-mSmo, ΔCRD-YFP-mSmo and ΔC-YFP-mSmo stably expressed in <italic>Smo</italic><sup>−/−</sup> cells and loaded on an 8% Tris-Glycine SDS-PAGE gel. The fraction of each protein with slower mobility on the gel was resistant to EndoH but sensitive to PGNaseF, suggesting post-Golgi localization. (<bold>D</bold>) 20(<italic>S</italic>)-OHC beads captured YFP-mSmo and ΔC-YFP-mSmo, but not ΔCRD-YFP-mSmo from lysates of cells stably expressing each protein. Binding to beads was not seen when 50 μM free 20(<italic>S</italic>)-OHC was added as a competitor.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.003">http://dx.doi.org/10.7554/eLife.01340.003</ext-link></p></caption><graphic xlink:href="elife01340f001"/></fig></p><p>A second binding site on Smo has been defined by side-chain oxysterols, oxidized derivatives of cholesterol carrying an additional hydroxyl group on the <italic>iso</italic>-octyl chain. Specific oxysterols can fully activate Hh signaling in the absence of Hh ligands in multiple cell types and also induce the accumulation of Smo in the primary cilium, a trafficking step essential for Smo to activate downstream signaling (<xref ref-type="bibr" rid="bib34">Kha et al., 2004</xref>; <xref ref-type="bibr" rid="bib17">Corcoran and Scott, 2006</xref>; <xref ref-type="bibr" rid="bib24">Dwyer et al., 2007</xref>; <xref ref-type="bibr" rid="bib37">Kim et al., 2007</xref>; <xref ref-type="bibr" rid="bib65">Rohatgi et al., 2007</xref>; <xref ref-type="bibr" rid="bib32">Johnson et al., 2011</xref>). We previously demonstrated that a specific side-chain oxysterol, 20(<italic>S</italic>)-hydroxycholesterol (20(<italic>S</italic>)-OHC), directly binds Smo in a manner that is highly stereospecific: the enantiomer, <italic>ent</italic>-20(<italic>S</italic>)-OHC, or the epimer, 20(<italic>R</italic>)-OHC, failed to bind Smo or to activate Hh signaling (<xref ref-type="bibr" rid="bib53">Nachtergaele et al., 2012</xref>). While this ‘oxysterol binding site’ showed allosteric interactions with the canonical cyclopamine binding site, it was clearly distinct since oxysterols did not show a competitive interaction with cyclopamine (<xref ref-type="bibr" rid="bib24">Dwyer et al., 2007</xref>; <xref ref-type="bibr" rid="bib53">Nachtergaele et al., 2012</xref>). Indeed, previous structural comparison studies have speculated that oxysterols bind to the extracellular CRD of Smo based on its relationship to the Fz CRD, which binds to the palmitoleyl group of secreted Wnt ligands (<xref ref-type="bibr" rid="bib6">Bazan and de Sauvage, 2009</xref>; <xref ref-type="bibr" rid="bib7">Bazan et al., 2012</xref>; <xref ref-type="bibr" rid="bib31">Janda et al., 2012</xref>; <xref ref-type="bibr" rid="bib71">Sharpe and de Sauvage, 2012</xref>). Wnt binding to the Fz CRD triggers signaling across the membrane, but the function of the Smo CRD has remained enigmatic.</p><p>We find that the extracellular CRD of Smo in vertebrates is both necessary and sufficient to bind to 20(<italic>S</italic>)-OHC, thus demonstrating that the cyclopamine and oxysterol binding sites occupy different domains in Smo. We determined the crystal structure of the zebrafish Smo CRD at 2.3 Å to provide a view of the oxysterol-binding pocket and to establish its relationship to the Fz CRD and other Fz-like CRDs that bind small hydrophobic ligands. Either deletion of the CRD or its inhibition by a new class of oxysterol-inspired small molecules can impair the signaling initiated by the native ligand Shh. Our results elucidate the molecular mechanism by which oxysterols activate Smo and show that the Smo CRD is a physiologically and therapeutically important target in the vertebrate Hh pathway.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>The extracellular CRD of mouse Smo is necessary and sufficient for binding to 20(<italic>S</italic>)-OHC</title><p>We previously developed a ligand affinity chromatography assay to measure the interaction between 20(<italic>S</italic>)-OHC and detergent-solubilized, full-length Smo (<xref ref-type="bibr" rid="bib53">Nachtergaele et al., 2012</xref>). For the studies presented here, we used a similar strategy to assay the interaction between truncated versions of Smo (<xref ref-type="fig" rid="fig1">Figure 1A</xref>) and 20(<italic>S</italic>)-OHC, using sepharose beads on which 20(<italic>S</italic>)-OHC was immobilized through an amino group installed on the <italic>iso</italic>-octyl chain (hereafter called 20(<italic>S</italic>)-OHC beads; <xref ref-type="fig" rid="fig1">Figure 1B</xref>). We produced deletion mutants (<xref ref-type="fig" rid="fig1">Figure 1A</xref>) of yellow fluorescent protein (YFP)-tagged mouse Smo (mSmo) lacking the CRD (ΔCRD-YFP-mSmo) or the C-terminal intracellular domain (ΔC-YFP-mSmo) and confirmed that these proteins were folded when stably expressed in Smo<sup>−/−</sup> mouse embryonic fibroblasts (MEFs) (<xref ref-type="bibr" rid="bib64">Rohatgi et al., 2009</xref>). Both truncated proteins demonstrated a slower migrating species that was resistant to Endoglycosidase H (EndoH), suggesting the presence of glycan modifications usually attached in the Golgi (<xref ref-type="fig" rid="fig1">Figure 1C</xref>) (<xref ref-type="bibr" rid="bib12">Chen et al., 2002a</xref>). For both YFP-mSmo and ΔC-YFP-mSmo, this post-Golgi band was selectively captured on 20(<italic>S</italic>)-OHC beads, showing that the C-terminal intracellular domain of Smo was dispensable for this interaction (<xref ref-type="fig" rid="fig1">Figure 1D</xref>). In this and subsequent experiments, specificity of binding was established by competition with free 20(<italic>S</italic>)-OHC. In contrast, ΔCRD-YFP-mSmo failed to show an interaction, suggesting that the CRD was required for oxysterol binding. Previous studies have shown that the truncated versions of Smo lacking either CRD or the C-terminal domain remain competent to bind cyclopamine and other cyclopamine-competitive ligands, consistent with these molecules interacting with the 7TM segment (<xref ref-type="bibr" rid="bib12">Chen et al., 2002a</xref>; <xref ref-type="bibr" rid="bib85">Wang et al., 2013</xref>). ΔCRD-YFP-mSmo also remained responsive to 7TM ligands (described below), confirming proper folding.<fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01340.004</object-id><label>Figure 2.</label><caption><title>The isolated mSmo CRD can bind oxysterols.</title><p>(<bold>A</bold>) Fractionation of the mSmo CRD-Fc protein on a Superose 6 gel-filtration column. The UV280 absorbance of each fraction (blue curve) is shown above the protein content of each fraction on a silver stained gel. Monodisperse protein (fractions 13–15) elutes in a sharp peak and binds to 20(<italic>S</italic>)-OHC beads (panels below), while aggregated protein runs as a broad peak (fractions 5–12) and fails to bind oxysterols. The indicated fractions (red boxes) were incubated with 20(<italic>S</italic>)-OHC beads in the presence or absence of free 20(<italic>S</italic>)-OHC competitor, and the amount of mSmo CRD-Fc protein captured on the beads or left in the flow through was assayed on an anti-Fc immunoblot. (<bold>B</bold>) A binding curve (K<sub><italic>d</italic></sub> ∼180 nM) for the mSmo CRD-Fc-20(<italic>S</italic>)-OHC interaction was measured by incubating a fixed amount of protein with increasing amounts of bead-immobilized sterol. (<bold>C</bold>) Binding of mSmo CRD-Fc to 20(<italic>S</italic>)-OHC beads is inhibited in a dose-responsive fashion by free 20(<italic>S</italic>)-OHC but not by the enantiomer <italic>ent</italic>-20(<italic>S</italic>)-OHC. A competition assay was used to test the ability of various oxysterols (<bold>D</bold>) or Smo ligands (<bold>E</bold>) to inhibit the binding of mSmo CRD-Fc to 20(<italic>S</italic>)-OHC beads. Anti-Fc immunoblots show the amount of protein in the input, captured on the beads, and left in the flow-through.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.004">http://dx.doi.org/10.7554/eLife.01340.004</ext-link></p></caption><graphic xlink:href="elife01340f002"/></fig></p><p>To determine if the mSmo CRD was sufficient to bind 20(<italic>S</italic>)-OHC, we purified isolated mSmo CRD fused to the constant region of the human IgG heavy chain (mSmo CRD-Fc; <xref ref-type="fig" rid="fig2">Figure 2A</xref>). The mSmo CRD-Fc protein secreted into the media of 293F cells ran as a smear on an SDS-PAGE gel. Further purification by Protein A affinity chromatography followed by gel filtration allowed us to isolate monodisperse mSmo CRD-Fc (<xref ref-type="fig" rid="fig2">Figure 2A</xref>, fractions 13–15). This well-behaved protein bound to 20(<italic>S</italic>)-OHC beads. A significant population of the protein was clearly misfolded, as it fractionated as a broad peak on a gel filtration column and failed to bind to 20(<italic>S</italic>)-OHC (<xref ref-type="fig" rid="fig2">Figure 2A</xref>, fractions 5–12). Binding of mSmo CRD-Fc to 20(<italic>S</italic>)-OHC beads was saturable (<xref ref-type="fig" rid="fig2">Figure 2B</xref>), specific (<xref ref-type="fig" rid="fig2">Figure 2C</xref>) and followed the same requirements for oxysterol stereochemistry and regiochemistry as previously described (<xref ref-type="fig" rid="fig2">Figure 2D</xref>) (<xref ref-type="bibr" rid="bib53">Nachtergaele et al., 2012</xref>). Binding could be inhibited by free 20(<italic>S</italic>)-OHC and free 20(<italic>S</italic>)-yne, the ∼10-fold more potent alkyne analog of 20(<italic>S</italic>)-OHC (<xref ref-type="bibr" rid="bib53">Nachtergaele et al., 2012</xref>). However, the enantiomer <italic>ent</italic>-20(<italic>S</italic>)-OHC, the epimer 20(<italic>R</italic>)-OHC, and 22(<italic>S</italic>)-OHC (all sterols that cannot activate Hh signaling) were unable to inhibit binding (<xref ref-type="bibr" rid="bib53">Nachtergaele et al., 2012</xref>). Ligands known to engage Smo at the cyclopamine binding site, SAG and SANT-1, failed to inhibit the binding of mSmo CRD-Fc to 20(<italic>S</italic>)-OHC beads, as did Itraconazole, a purported Smo ligand that binds to an unknown site (<xref ref-type="fig" rid="fig2">Figure 2E</xref>) (<xref ref-type="bibr" rid="bib12">Chen et al., 2002a</xref>, <xref ref-type="bibr" rid="bib13">2002b</xref>; <xref ref-type="bibr" rid="bib35">Kim et al., 2010</xref>). While our manuscript was in preparation, an independent study also reported the interaction between oxysterols and the Smo CRD (<xref ref-type="bibr" rid="bib55">Nedelcu et al., 2013</xref>). Overall, our results show that the cyclopamine and oxysterol binding sites on Smo are distinct. For clarity, we hereafter refer to these sites as the 7TM and CRD sites, respectively.</p></sec><sec id="s2-2"><title>The Smo CRD is required for Shh-induced signaling</title><p>To investigate the function of the Smo CRD for signaling induced by the native ligand Shh, YFP-tagged mSmo variants were expressed by stable retroviral transduction in Smo<sup>−/−</sup> MEFs to avoid the confounding effects of endogenous Smo. These clonal Smo<sup>−/−</sup>:YFP-mSmo cells could activate a Hh target gene, Gli1, when exposed to Shh (which binds and inactivates Ptch1) or to the Smo agonists SAG and 20(<italic>S</italic>)-OHC, which bind to the 7TM and CRD sites, respectively (<xref ref-type="fig" rid="fig3">Figure 3A</xref>) (<xref ref-type="bibr" rid="bib64">Rohatgi et al., 2009</xref>). In an independent, non-transcriptional measure of signaling, loss of the repressor form of Gli3 (Gli3R) was observed in response to all three agonists (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). In contrast, ΔCRD-YFP-mSmo failed to activate Hh target genes or to extinguish Gli3R levels in response to both Shh and 20(<italic>S</italic>)-OHC, but retained its ability to respond to SAG (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). Identical results were obtained using a luciferase-based Hh reporter transiently expressed along with YFP-mSmo or ΔCRD-YFP-mSmo in Smo<sup>−/−</sup> cells (<xref ref-type="fig" rid="fig3">Figure 3B</xref>) (<xref ref-type="bibr" rid="bib68">Sasaki et al., 1997</xref>; <xref ref-type="bibr" rid="bib82">Varjosalo et al., 2006</xref>). SAG activated ΔCRD-YFP-mSmo remained susceptible to inhibition by cyclopamine, consistent with an intact 7TM site (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). As noted previously, ΔCRD-YFP-mSmo was not constitutively active, but it did demonstrate a higher level of basal activity in Hh reporter assays (<xref ref-type="bibr" rid="bib79">Taipale et al., 2002</xref>; <xref ref-type="bibr" rid="bib1">Aanstad et al., 2009</xref>). The SAG responsiveness shows that ΔCRD-YFP-mSmo is not a misfolded or inactive protein; instead, it supports the notion that the CRD of Smo mediates the response to oxysterols while the 7TM segment mediates the response to SAG. Most significantly, this result suggests that the CRD plays an important role in mediating the response to Shh and thus in mediating the interaction between Ptch1 and Smo.<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01340.005</object-id><label>Figure 3.</label><caption><title>The mSmo CRD is required for Shh- and oxysterol-mediated activation of Hh signaling.</title><p>(<bold>A</bold>) Smo<sup>−/−</sup> cells stably expressing full-length (FL) YFP-mSmo or ΔCRD-YFP-mSmo were treated with Shh, SAG (100 nM) or 20(<italic>S</italic>)-OHC (10 μM). Levels of Gli1 and Gli3R protein, determined by immunoblotting after fractionation on an 8% Tris-glycine SDS-PAGE gel, were taken as a metric of pathway activation. An anti-YFP blot shows the levels of YFP-mSmo in each sample, and p38 levels are used as a loading control. (<bold>B</bold> and <bold>C</bold>) A luciferase-based Hh reporter gene was used to measure signaling in Smo<sup>−/−</sup> cells transiently transfected with constructs encoding YFP-mSmo or ΔCRD-YFP-mSmo and then treated with the indicated Smo ligands. In (<bold>C</bold>), ΔCRD-YFP-mSmo activated with SAG (25 nM) can be inhibited by the co-administration of cyclopamine (5 μM). Error bars denote S.D. (n = 3).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.005">http://dx.doi.org/10.7554/eLife.01340.005</ext-link></p></caption><graphic xlink:href="elife01340f003"/></fig></p></sec><sec id="s2-3"><title>The oxysterol–CRD interaction is conserved across vertebrates</title><p>We tested the binding of Smo from various species to 20(<italic>S</italic>)-OHC beads (<xref ref-type="fig" rid="fig4">Figure 4A,B</xref>). Both full-length <italic>Drosophila melanogaster</italic> Smo (dSmo) and the isolated dSmo CRD failed to bind 20(<italic>S</italic>)-OHC beads. However, a truncated version of zebrafish Smo (zSmo) lacking the intracellular C-terminal region (YFP-zSmoΔC), expressed in mammalian cells and solubilized with detergent, bound to 20(<italic>S</italic>)-OHC beads, showing that this interaction is likely conserved in the vertebrate (but not in the invertebrate) Hh pathway.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01340.006</object-id><label>Figure 4.</label><caption><title>The Smo-oxysterol interaction is conserved in vertebrates.</title><p>(<bold>A</bold>) The interaction of 20(<italic>S</italic>)-OHC beads with full-length mSmo, full-length Drosophila Smo (dSmo) or zebrafish Smo (zSmo) carrying a truncation of the intracellular C-terminal tail (zSmoΔC) was tested in the presence of free 20(<italic>S</italic>)-OHC or its enantiomer. (<bold>B</bold>) The zSmo ectodomain (which includes the CRD) can bind to 20(<italic>S</italic>)-OHC beads, but the dSmo CRD cannot. (<bold>C</bold>) Zebrafish embryos (30hpf) carrying a GFP transgene driven by the <italic>engrailed2a</italic> promoter were treated with 20(<italic>S</italic>)-OHC (50 µM) or cyclopamine (40 µM) and assessed for GFP expression by fluorescence and <italic>ptch2</italic> expression by in situ hybridization. See <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref> for quantitation. (<bold>D</bold>) A binding curve (K<sub><italic>d</italic></sub> ∼170 nM) for the zSmo ectodomain-20(<italic>S</italic>)-OHC interaction was measured by incubating a fixed amount of protein with increasing amounts of bead-immobilized sterol. The amount of zSmo ectodomain captured on the beads (shown in the graph) was quantitated from a coomassie-stained SDS-PAGE gel shown above. (<bold>E</bold>) Binding of the zSmo ectodomain to 20(<italic>S</italic>)-OHC beads was inhibited in a dose-responsive fashion by free 20(<italic>S</italic>)-OHC but not by its enantiomer. (<bold>F</bold> and <bold>G</bold>) Coomassie-stained SDS-PAGE gels show the amount of zSmo ectodomain captured on 20(<italic>S</italic>)-OHC beads in the presence of various oxysterols (<bold>F</bold>) or Smo ligands (<bold>G</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.006">http://dx.doi.org/10.7554/eLife.01340.006</ext-link></p></caption><graphic xlink:href="elife01340f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01340.007</object-id><label>Figure 4—Figure supplement 1.</label><caption><title>20(<italic>S</italic>)-OHC activates Hedgehog signaling in zebrafish embryos.</title><p>Zebrafish embryos (30hpf) carrying a GFP transgene driven by the <italic>engrailed2a</italic> (eng2a:GFP) promoter were treated with 20(<italic>S</italic>)-OHC (50 µM) or cyclopamine (40 µM) and assessed for GFP expression by fluorescence. To quantify the effect of each treatment on eng2a:GFP expression, the width of the GFP-positive domain was measured at three points along the length of each embryo (right panel, white lines) and averaged. The average width is plotted as a scatter plot, with each point representing one embryo. 12–16 embryos per condition are depicted. Red bars represent mean ± SD. All conditions were significantly different from each other (****p<0.0001, one-way ANOVA with Bonferroni correction for multiple comparisons.)</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.007">http://dx.doi.org/10.7554/eLife.01340.007</ext-link></p></caption><graphic xlink:href="elife01340fs001"/></fig></fig-group></p><p>We tested whether the zebrafish Hh pathway was responsive to oxysterols, because our structural studies described below focused on Smo protein from this species. Full-length zebrafish Smo was poorly expressed in mammalian cells, precluding tests of its responsiveness to oxysterols in cultured cells. Hh pathway activity underlies the specification of distinct muscle cell types in the zebrafish embryo, in part through the activation of the <italic>engrailed2</italic> (<italic>eng2</italic>) gene in subsets of slow-twitch and fast-twitch myoblasts (<xref ref-type="bibr" rid="bib90">Wolff et al., 2003</xref>). To investigate the in vivo significance of the interaction between 20(<italic>S</italic>)-OHC and Smo, we treated embryos carrying an eng2a:GFP reporter construct (<xref ref-type="bibr" rid="bib43">Maurya et al., 2011</xref>) with either 20(<italic>S</italic>)-OHC or cyclopamine. As expected, cyclopamine treatment suppressed expression of the reporter gene; by contrast, 20(<italic>S</italic>)-OHC treated embryos showed a significant increase in the number of GFP positive fast twitch muscles compared to vehicle treated embryos (<xref ref-type="fig" rid="fig4">Figure 4C</xref> and <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). Consistent with this, 20(<italic>S</italic>)-OHC treated embryos also showed a modest expansion of the expression domain of the endogenous Hh target gene, <italic>ptch2</italic>. These data show that 20(<italic>S</italic>)-OHC can induce Hh signaling in the context of a living vertebrate embryo and suggest that the in vitro interaction between zebrafish Smo and 20(<italic>S</italic>)-OHC induces its activation in vivo.</p><p>We succeeded in purifying large quantities of the zSmo ectodomain, encompassing both the CRD and the segment between the CRD and the first transmembrane helix. The zSmo ectodomain demonstrated saturable, specific binding to 20(<italic>S</italic>)-OHC beads (<xref ref-type="fig" rid="fig4">Figure 4D,E</xref>). Similar to the mouse protein, binding could be inhibited by oxysterols that activate Hh signaling but not by those that cannot (<xref ref-type="fig" rid="fig4">Figure 4F</xref>); 7TM site ligands also failed to compete for binding (<xref ref-type="fig" rid="fig4">Figure 4G</xref>).</p></sec><sec id="s2-4"><title>The structure of the Smo CRD from zebrafish</title><p>To obtain molecular insights into the architecture of the Smo extracellular region, we crystallized the zSmo ectodomain and determined its structure using selenomethionine-labeled protein for phasing (<xref ref-type="table" rid="tbl1">Table 1</xref>, <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1A</xref>). Refinement resulted in an R-factor of 21.6% (R-free: 26.0%) with two zSmo molecules in the crystallographic asymmetric unit, each composed of a well-defined model that included residues 41–158 (root mean square deviation [RMSD]: 0.60 Å for 118 Cα positions, <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1B</xref>). Although we set-up crystallization trials with the entire zSmo ectodomain (residues 29–212), the N- and C-terminal regions could not be traced due to missing electron density and thus were not included in the final model (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref>). The portion of the zSmo ectodomain spanning residues 41–158 (visible in our structure) shows sequence similarity to the previously identified CRD in the Fz protein family (<xref ref-type="bibr" rid="bib18">Dann et al., 2001</xref>) and thus will hereafter be called the zSmo CRD.<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01340.008</object-id><label>Table 1.</label><caption><p>Data collection and refinement statistics</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.008">http://dx.doi.org/10.7554/eLife.01340.008</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th/><th>SeMet-substituted zSmo CRD</th><th>Native zSmo CRD</th></tr></thead><tbody><tr><td colspan="3">Data collection</td></tr><tr><td> Beamline</td><td>ESRF-ID23-EH1</td><td>DIAMOND I03</td></tr><tr><td> Wavelength</td><td>0.979</td><td>1.000</td></tr><tr><td> Space group</td><td>P4<sub>3</sub>2<sub>1</sub>2</td><td>P4<sub>3</sub>2<sub>1</sub>2</td></tr><tr><td> Cell Dimension (Å)</td><td>a, b = 68.2; c = 92.3</td><td>a, b = 68.6; c = 95.3</td></tr><tr><td> Resolution</td><td>48.0–2.3 (2.36-2.30)</td><td>31.0–2.6 (2.67-2.60)</td></tr><tr><td> Completeness (%)</td><td>99.2 (92.8)</td><td>98.9 (94.7)</td></tr><tr><td> Unique reflections</td><td>10,085</td><td>7307 (491)</td></tr><tr><td> R<sub>merge</sub> (%)</td><td>10.0 (92.0)</td><td>13.4 (64.8)</td></tr><tr><td> I/σ(I)</td><td>25.5 (3.9)</td><td>14.4 (2.4)</td></tr><tr><td> Multiplicity</td><td>26.5 (23.7)</td><td>8.8 (6.0)</td></tr><tr><td colspan="3">Refinement</td></tr><tr><td> Resolution range (Å)</td><td>30.50–2.30</td><td>31.00–2.60</td></tr><tr><td> No. reflections</td><td>9576</td><td>7275</td></tr><tr><td> <italic>R</italic><sub>work</sub> (%)</td><td>23.6</td><td>21.6</td></tr><tr><td> <italic>R</italic><sub>free</sub> (%)</td><td>28.4</td><td>26.0</td></tr><tr><td> No. atoms (protein/Zn/Na/water)</td><td>1880/1/3/28</td><td>1880/1/2/48</td></tr><tr><td> B-factors (Å<sup>2</sup>) (protein/Zn/Na/water)</td><td>57/60/43/48</td><td>40/32/27/30</td></tr><tr><td> r.m.s. deviations</td><td/><td/></tr><tr><td> Bond lengths (Å)</td><td>0.012</td><td>0.004</td></tr><tr><td> Bond angles (°)</td><td>1.604</td><td>0.714</td></tr><tr><td> Ramachandran statistics</td><td/><td/></tr><tr><td> Favored (%)</td><td>96.5</td><td>97.9</td></tr><tr><td> Disallowed (%)</td><td>0.4</td><td>0</td></tr></tbody></table><table-wrap-foot><fn><p>Each structure was determined from one crystal. Numbers in parentheses refer to the highest resolution shell. <italic>R</italic><sub>free</sub> equals the <italic>R</italic>-factor against 5% of the data.</p></fn></table-wrap-foot></table-wrap></p><p>The small interface between the two zSmo CRD molecules observed in the asymmetric unit of the crystal (buried surface area of 490 Å<sup>2</sup>) and a crystal contact formed by a zinc ion bonded to three different protein chains (one chain A and two chain B molecules; <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1C</xref>) suggested that the dimeric arrangement observed in the crystal is not likely to be of functional significance. In agreement with this crystal packing analysis, purified zSmo ectodomain behaved as a monomer in solution at low concentration (5 µM) when assessed using multi angle light scattering (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1D</xref>).</p><p>The zSmo CRD monomer adopts a globular fold composed of four α helices (α1: residues Q77-N92; α2: P94-Y108; α3: Q122-N130; α3′: S133-E138) and a short two-stranded β sheet (β1: K43-S45 and β2: K116-E118; <xref ref-type="fig" rid="fig5">Figure 5A</xref> and <xref ref-type="fig" rid="fig5s2">Figure 5—figure Supplement 2</xref>). This arrangement is stabilized by five disulfide bridges (labeled *, I, II, III, IV in <xref ref-type="fig" rid="fig5">Figure 5A</xref>). Disulfide bridges I, II, III, and IV lock the four helices together into a tight bundle, whereas disulfide bridge *, formed by the N- and C-terminal cysteines, orients the termini in close proximity and away from the helical bundle (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). Structure-based evolutionary analysis of zSmo CRD revealed that the closest structural relatives are the CRDs of Frizzled 8 (Fz8; <xref ref-type="bibr" rid="bib18">Dann et al., 2001</xref>; <xref ref-type="bibr" rid="bib31">Janda et al., 2012</xref>), secreted Frizzled-related protein 3 (sFRP3, <xref ref-type="bibr" rid="bib18">Dann et al., 2001</xref>) and muscle-specific kinase (MuSK, <xref ref-type="bibr" rid="bib74">Stiegler et al., 2009</xref>), shown clustered in the blue branch in <xref ref-type="fig" rid="fig5">Figure 5B</xref> (<xref ref-type="fig" rid="fig5s3">Figure 5—figure supplement 3</xref>). These three structures show a similar helical bundle arrangement compared to the zSmo CRD, with the exception of a rearrangement of helix α3 and α3′, which forms a continuous helix in Fz8 and sFRP3. Strikingly, 4 out of 5 disulfide bridges are highly conserved (I, II, III, and IV), retaining the overall fold of the helix bundle. Only one disulfide bridge (labeled with an asterisk * in <xref ref-type="fig" rid="fig5">Figure 5A</xref>) is not conserved, resulting in a rearrangement of the relative orientations of the two termini compared to the zSmo CRD (<xref ref-type="fig" rid="fig5">Figure 5C–E</xref>).<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01340.009</object-id><label>Figure 5.</label><caption><title>Structural analysis of the zebrafish Smo CRD.</title><p>(<bold>A</bold>) Ribbon diagram of zSmo CRD in rainbow coloring from blue (N-terminus) to red (C-terminus) with the secondary structure elements numbered. The four disulfide bridges (black sticks) conserved in all Fz-like CRDs are depicted with Roman numerals, and the non-conserved disulfide bridge is marked with an asterisk (*). N- and C-termini are labeled. (<bold>B</bold>) Structural phylogenetic analysis of the CRDs. Structural superposition of CRDs from zSmo, Frizzled 8 (Fz8, PDB ID 4F0A, <xref ref-type="bibr" rid="bib31">Janda et al., 2012</xref>), secreted Frizzled-related protein 3 (sFRP3, PDB ID 1IJX, <xref ref-type="bibr" rid="bib18">Dann et al., 2001</xref>), muscle-specific kinase (MuSK, PDB ID 3HKL, <xref ref-type="bibr" rid="bib74">Stiegler et al., 2009</xref>), Niemann-Pick C1 protein (NPC1, PDB ID 3GKI, <xref ref-type="bibr" rid="bib38">Kwon et al., 2009</xref>), riboflavin-binding protein (RFBP, <xref ref-type="bibr" rid="bib49">Monaco, 1997</xref>), and folate receptor α (FRα, PDB ID 4LRH, <xref ref-type="bibr" rid="bib11">Chen et al., 2013</xref>) were superimposed using SHP (<xref ref-type="bibr" rid="bib76">Stuart et al., 1979</xref>; <xref ref-type="bibr" rid="bib62">Riffel et al., 2002</xref>). CRDs that form ligand-binding pockets (red background) or grooves (blue background) form two distinct evolutionary branches. In addition, CRDs show distant structural similarity to the extracellular domains of glypicans (<xref ref-type="bibr" rid="bib58">Pei and Grishin, 2012</xref>). However, analysis of the crystal structures of glypicans Dally-like protein and glypican 1 revealed no apparent grooves or pockets that could accommodate small molecules (<xref ref-type="bibr" rid="bib36">Kim et al., 2011</xref>; <xref ref-type="bibr" rid="bib77">Svensson et al., 2012</xref>) and thus were not included in our structural analyses. (<bold>C</bold>–<bold>H</bold>) Ribbon diagrams of superimposed Fz-like CRD domains from the structural phylogenetic analysis in (<bold>B</bold>). (<bold>C</bold>) Fz8-palmitoleyl complex, (<bold>D</bold>) sFRP3, (<bold>E</bold>) MuSK, (<bold>F</bold>) NPC1-cholesterol complex, (<bold>G</bold>) RFBP-riboflavin complex, (<bold>H</bold>) FRα-folate complex. Color coding and labeling follows (<bold>A</bold>). Ligands are shown as spheres in atomic coloring (carbon: slate; oxygen: red; nitrogen: blue). In (<bold>F</bold>–<bold>H</bold>) the conserved disulfide bridges are highlighted with a circle. NPC1 (<bold>F</bold>) does not contain disufide bridge IV.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.009">http://dx.doi.org/10.7554/eLife.01340.009</ext-link></p></caption><graphic xlink:href="elife01340f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01340.010</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Electron density of the zSmo CRD structure and oligomeric state of the zSmo ectodomain.</title><p>(<bold>A</bold>) SeMet SAD-phased and density modified map from RESOLVE (<xref ref-type="bibr" rid="bib81">Terwilliger, 2003</xref>) calculated to 2.3 Å resolution and contoured at 1.0 σ showing the two core zSmo CRD helices α1 and α2. (<bold>B</bold>) SigmaA-weighted <italic>2F</italic><sub><italic>O</italic></sub><italic>-F</italic><sub><italic>C</italic></sub> map of the final model of SeMet-labeled zSmo ectodomain from REFMAC (<xref ref-type="bibr" rid="bib52">Murshudov et al., 1997</xref>) at 2.3 Å resolution and contoured at 1.0 σ. View is the same as in (<bold>A</bold>). (<bold>C</bold>) Close-up view of the zinc-binding site in the zSmo CRD crystal structure. The anomalous difference Fourier map (yellow, contoured at 5 σ) and SigmaA-weighted <italic>2F</italic><sub><italic>O</italic></sub><italic>-F</italic><sub><italic>C</italic></sub> map (blue, contoured at 1.0 σ) of the final model of native zSmo CRD were calculated to 2.6 Å. Note that zinc is present in a crystal contact formed by three different zSmo chains. (<bold>D</bold>) Multi angle light scattering of the glycosylated zSmo ectodomain (expressed in mammalian cells) indicates a molecular mass (red scattered dots) of 24.43 ± 0.9 kDa and is in agreement with the theoretical molecular mass for a non-glycosylated monomer (20.4 kDa). The zSmo ectodomain has two predicted N-linked glycosylation sites (each accounting for 2 kDa), which explains the difference between the theoretical and MALS-derived molecular mass. Protein concentration at the elution peak was 8.123×10<sup>−5</sup> g/ml.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.010">http://dx.doi.org/10.7554/eLife.01340.010</ext-link></p></caption><graphic xlink:href="elife01340fs002"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01340.011</object-id><label>Figure 5—figure supplement 2.</label><caption><title>Sequence alignment of the ectodomains of Smo family members and the CRD of mFz8.</title><p>Sequences were aligned using ClustalW (<xref ref-type="bibr" rid="bib39">Larkin et al., 2007</xref>) and adjusted manually for mFz8. Secondary structure assignments of zSmo CRD and mFz8 (PDB ID 4F0A, <xref ref-type="bibr" rid="bib31">Janda et al., 2012</xref>) are displayed above the alignment and color-coded as in <xref ref-type="fig" rid="fig5">Figure 5</xref>. Disulfide bonds are highlighted and numbered as in <xref ref-type="fig" rid="fig5">Figure 5A</xref>. Smo disulfide bond *, which is not conserved in the CRD protein family, is marked in yellow. The two cysteine residues of mFz8 forming the rearranged disulfide bond (marked with * in <xref ref-type="fig" rid="fig5">Figure 5C</xref>) are highlighted in violet. The box indicates the zSmo residues visible in our crystal structure. Residues lining the oxysterol binding groove in Smo are highlighted in red for zSmo and residues lining the palmitoleyl-binding groove in mFz8 are in blue. Mutated mSmo residues that substantially reduced binding to 20(<italic>S</italic>)-OHC beads are depicted with a plus (+) below the alignment. Mutated residues that did not reduce binding are marked with a number sign (#).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.011">http://dx.doi.org/10.7554/eLife.01340.011</ext-link></p></caption><graphic xlink:href="elife01340fs003"/></fig><fig id="fig5s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01340.012</object-id><label>Figure 5—figure supplement 3.</label><caption><title>Structural comparison of CRDs.</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.012">http://dx.doi.org/10.7554/eLife.01340.012</ext-link></p></caption><graphic xlink:href="elife01340fs004"/></fig></fig-group></p><p>Using structure fold recognition methods, Bazan and de Sauvage identified an additional group of Fz-like CRD containing proteins (<xref ref-type="bibr" rid="bib6">Bazan and de Sauvage, 2009</xref>). These include the Niemann-Pick C1 protein (NPC1) and the riboflavin-binding protein (RFBP). Our evolutionary structural analysis confirmed their findings and allowed us to add the folate receptor α (FRα) to this group (<xref ref-type="fig" rid="fig5">Figure 5B</xref>, red branch and <xref ref-type="fig" rid="fig5s3">Figure 5—figure supplement 3</xref>) (<xref ref-type="bibr" rid="bib11">Chen et al., 2013</xref>). Structural comparison of these proteins to the zSmo CRD revealed the common features identified for Fz-like CRDs, namely the helical bundle (formed by helices α1, α2 and α3) and the four conserved disulfide bonds that stabilize the fold and the relative orientations of the helices (<xref ref-type="fig" rid="fig5">Figure 5F–H</xref>).</p></sec><sec id="s2-5"><title>Mapping the Smo oxysterol binding site</title><p>A common feature of the Fz-like CRD family members is their ability to bind small, hydrophobic molecules in a pocket formed by the core helices α1, α2 and α3. While NPC1, RFBP and FRα bury their respective ligands in the protein core (cholesterol in NPC1, riboflavin in RFBP and folate in FRα) with the help of extensive protrusions from the core CRD fold (shown in gray in <xref ref-type="fig" rid="fig5">Figure 5F–H</xref>), Fz8, the closest structural homolog of the zSmo CRD structure, binds the palmitoleyl moiety covalently linked to Wnt proteins in a shallow groove (<xref ref-type="fig" rid="fig5">Figure 5C</xref>; <xref ref-type="bibr" rid="bib31">Janda et al., 2012</xref>). To investigate the putative oxysterol binding site in the Smo CRD, we calculated the volumes of potential binding pockets in our zSmo CRD structure. The most prominent groove is indeed located at an equivalent position to the Fz8 palmitoleyl-binding groove (<xref ref-type="fig" rid="fig6">Figure 6A,B</xref>). The residues forming this groove are highly conserved in all vertebrate Smo CRDs (<xref ref-type="fig" rid="fig6">Figure 6C</xref> and <xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref>), and the volume (551 Å<sup>3</sup>) and shape of the groove is sufficient for 20(<italic>S</italic>)-OHC binding. Computational docking using AutoDock (<xref ref-type="bibr" rid="bib50">Morris et al., 2009</xref>) showed that the hydrophobic groove on the zSmo CRD surface (<xref ref-type="fig" rid="fig6">Figure 6A</xref>) can accommodate 20(<italic>S</italic>)-OHC with a favorable free energy of binding (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1A–C</xref>). The four rings of the oxysterol are predicted to lie on the base of the groove lined with zSmo residues W87 and L90 and make additional potential hydrophobic interactions with zSmo residues M86, G89, Y108, G140, P142 and F144.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.01340.013</object-id><label>Figure 6.</label><caption><title>Mapping and analysis of the zSmo oxysterol binding site.</title><p>(<bold>A</bold> and <bold>B</bold>) Ribbon representations of the zSmo CRD (<bold>A</bold>) and the Fz8 CRD-palmitoleyl (<bold>B</bold>) structures. View and presentation follows <xref ref-type="fig" rid="fig5">Figure 5A</xref>. The palmitoleyl-binding pocket of Fz8 is depicted as a cyan wire mesh and the corresponding pocket in the zSmo CRD structure is in red wire mesh. Volumes were calculated using the program Volumes (RE Esnouf, unpublished), with a 1.4 Å probe radius. The palmitoleyl moiety is shown as slate spheres. (<bold>C</bold>) The solvent accessible surface of the zSmo CRD is color-coded according to residue conservation (from non-conserved, white, to conserved, black) based on alignments containing amino acid sequences from >80 vertebrate Smo proteins. The right panel is rotated 90° around the y-axis relative to the left panel. Residues on the opposite face of the oxysterol-binding pocket that were subjected to mutagenesis are labeled. (<bold>D</bold>) Close-up view of the potential 20(<italic>S</italic>)-OHC binding site in the zSmo CRD structure. Residues predicted to make contacts with 20(<italic>S</italic>)-OHC are shown in stick representation and highlighted in red. Boundaries of the hydrophobic groove are marked with dotted lines. zSmo residues are numbered, with the corresponding mSmo residues in parentheses. (<bold>E</bold>) The indicated full-length mSmo point mutants were tested for their interaction with 20(<italic>S</italic>)-OHC beads in the absence or presence of free 20(<italic>S</italic>)-OHC competitor. Well-folded Smo mutants ran as a double band on a 4–12% Bis-Tris gradient gel (arrowheads), with only the slower-migrating species being captured on 20(<italic>S</italic>)-OHC beads. (<bold>F</bold>) A Hh reporter assay was used to measure signaling in Smo<sup>−/−</sup> cells transiently transfected with constructs encoding various mSmo point mutants and then treated (48 hr) with Shh, SAG (100 nM) or 20(<italic>S</italic>)-OHC (10 μM). The maximum reporter response for each mutant was set to 100%. Error bars denote S.D. (n = 3).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.013">http://dx.doi.org/10.7554/eLife.01340.013</ext-link></p></caption><graphic xlink:href="elife01340f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01340.014</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Molecular modeling analysis of the zSmo CRD.</title><p>(<bold>A</bold>) Close-up view of the Smo oxysterol-binding groove. Presentation is as in <xref ref-type="fig" rid="fig6">Figure 6D</xref>. Boundaries of the potential binding site are marked with dashed lines. (<bold>B</bold>) The computationally docked structure of 20(<italic>S</italic>)-OHC (stick representation; carbon: orange, oxygen: red) in complex with the zSmo CRD suggests energetically favorable interactions between the two molecules with an estimated free binding energy of −9.0 kcal/mol and estimated inhibition constant, K<sub>i</sub>, equal to 260 nM. View is as in (<bold>A</bold>). (<bold>C</bold>) Close-up view of the palmitoyl binding site in the Fz8-Wnt complex (PDB ID 4F0A, <xref ref-type="bibr" rid="bib31">Janda et al., 2012</xref>) in the same orientation as in (<bold>A</bold>). (<bold>D</bold>) Homology model of the Drosophila Smo-CRD (dSmo) based on the zSmo structure (sequence identity: 42%) reveals a substantially wider groove compared to grooves of the CRDs from zSmo and Fz8. Three key residues (Met86, Tyr108, Gly140) are absent in dSmo that have been shown to be essential for 20(<italic>S</italic>)-OHC binding to vertebrate Smo.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.014">http://dx.doi.org/10.7554/eLife.01340.014</ext-link></p></caption><graphic xlink:href="elife01340fs005"/></fig><fig id="fig6s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01340.015</object-id><label>Figure 6—figure supplement 2.</label><caption><title>Mutagenesis of the putative oxysterol binding site in the mSmo CRD.</title><p>(<bold>A</bold>) The indicated full-length mSmo point mutants were tested for their interaction with 20(<italic>S</italic>)-OHC beads in the absence or presence of free 20(<italic>S</italic>)-OHC competitor. (<bold>B</bold>) Heat map summarizing the signaling properties of all Smo mutants tested in this study in response to Shh, SAG and 20(<italic>S</italic>)-OHC, using assays of the type shown in <xref ref-type="fig" rid="fig6">Figure 6E,F</xref>. Mutants that were not responsive to SAG and/or ran as a single band on an SDS-PAGE gel (indicating lack of glycan chains attached in the Golgi apparatus) were deemed not folded (NF). Mutants were only assessed for Shh and 20(<italic>S</italic>)-OHC responsiveness if they had SAG responsiveness that was at least 75% of that seen with wild-type mSmo.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.015">http://dx.doi.org/10.7554/eLife.01340.015</ext-link></p></caption><graphic xlink:href="elife01340fs006"/></fig></fig-group></p><p>To test this model for the oxysterol-binding pocket, we mutated Smo residues that map to this pocket and, as controls, other residues that point away from the pocket or that are on the opposite face of the molecule (<xref ref-type="fig" rid="fig6">Figure 6C,D</xref>). All mutations were made in full-length mouse Smo, and mutant proteins were tested for binding to 20(<italic>S</italic>)-OHC beads after detergent-solubilization from membranes (<xref ref-type="fig" rid="fig6">Figure 6E</xref>). <xref ref-type="fig" rid="fig6">Figure 6D</xref> shows corresponding mouse and zebrafish residue numbers, and hereafter the residues are numbered according to the mouse sequence. Only those mutants that fractionated as a doublet on an SDS-PAGE gel were evaluated because this property demonstrates post-Golgi trafficking and hence correct folding (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). Mutations in residues on the opposite face of the putative sterol binding pocket (E162A, P120A/E/G, P128S/E/R, P88N, L150A/D/S) or at the periphery of the pocket (R165A/E and N118A) did not disrupt binding to 20(<italic>S</italic>)-OHC beads (<xref ref-type="fig" rid="fig6">Figure 6E</xref> and <xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2</xref>). In contrast, mutations in residues that frame the putative oxysterol pocket (L112A, L112D, G115F, L116A, Y134F, G166F, P168A, F170A) substantially reduced binding to 20(<italic>S</italic>)-OHC beads (<xref ref-type="fig" rid="fig6">Figure 6E</xref> and <xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2</xref>). Taken together, our mutagenesis data support the structure-based model for the interaction between oxysterols and the Smo CRD.</p><p>To understand why <italic>Drosophila</italic> Smo does not bind oxysterols, we constructed a homology model of the dSmo CRD based on the zSmo structure (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1D</xref>). Despite the notable sequence identity between zebrafish and <italic>Drosophila</italic> Smo CRDs (∼42%) and the conserved disulfide bond pattern, the homology model revealed a substantially different oxysterol-binding groove on the dSmo CRD surface. 5 out of 8 residues that are essential for vertebrate Smo interactions with oxysterols (zSmo residues M86, W87, G89, Y108 and G140) are different in dSmo (corresponding dSmo residues D129, Y130, A132, F151 and F187; <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1D</xref>), potentially providing an explanation for why dSmo does not bind to oxysterols.</p><p>Finally, we tested a subset of these mSmo mutants for their ability to rescue Hh signaling in Smo<sup>−/−</sup> cells treated with Shh, SAG or 20(<italic>S</italic>)-OHC. The mutations that preserved 20(<italic>S</italic>)-OHC binding also preserved mSmo responsiveness to all three agonists (<xref ref-type="fig" rid="fig6">Figure 6F</xref> and <xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2B</xref>). The most informative mutations were G115F, P168A and Y134F, the last a conservative change that substitutes a <italic>Drosophila</italic> residue (F) for the corresponding mouse residue (Y). All three mutants were responsive to SAG, showing that they were not disabled, but demonstrated substantially reduced 20(<italic>S</italic>)-OHC binding and responsiveness, with the mSmo Y134F being completely unresponsive (<xref ref-type="fig" rid="fig6">Figure 6F</xref>). Interestingly, Shh-responsiveness was unaffected in mSmo G115F but significantly reduced in both mSmo Y134F and P168A. Finally, there were a few mutants (e.g., L116A) that did not show strong binding to 20(<italic>S</italic>)-OHC beads in our in vitro assay but still modestly responded to 20(<italic>S</italic>)-OHC when introduced into Smo<sup>−/−</sup> cells. This discrepancy may be due to the fact that our signaling assay in intact cells is more sensitive than the binding assay with solubilized proteins, which is conducted in the presence of high detergent to maintain Smo solubility after extraction from membranes.</p></sec><sec id="s2-6"><title>Oxysterol-based inhibitors that target the Smo CRD</title><p>The current generation Smo inhibitors that have entered the clinic, including the FDA-approved drug Vismodegib, all engage the 7TM site on Smo (<xref ref-type="bibr" rid="bib29">Frank-Kamenetsky et al., 2002</xref>). However, mutations that prevent drug binding or drug activity can lead to clinically relevant resistance to these agents (<xref ref-type="bibr" rid="bib91">Yauch et al., 2009</xref>; <xref ref-type="bibr" rid="bib23">Dijkgraaf et al., 2011</xref>). Antagonists that engage the oxysterol binding site in the CRD would represent an orthogonal strategy for Smo inhibition.</p><p>To design such inhibitors, we considered two observations from our prior structure–activity relationship (SAR) studies on 20(<italic>S</italic>)-OHC (<xref ref-type="bibr" rid="bib53">Nachtergaele et al., 2012</xref>). First, the stereochemistry at position 20 that determines the spatial relationship between the ring system and the <italic>iso</italic>-octyl chain is critical for the ability of 20(<italic>S</italic>)-OHC to activate Smo, since 20(<italic>R</italic>)-OHC is inactive. Second, the replacement of the <italic>iso</italic>-butyl group at the end of the <italic>iso</italic>-octyl chain with an alkyne group increased Hh-activation potency by ∼10-fold (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). Starting from this high-potency Smo activator 20(<italic>S</italic>)-yne, we inverted the stereochemistry at position 20 to make 20(<italic>R</italic>)-yne or oxidized the hydroxyl group to a ketone, changing carbon 20 to a planar <italic>sp</italic><sup>2</sup> hybridized center, to make 20-keto-yne (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). Both molecules blocked the binding of mSmo CRD-Fc to 20(<italic>S</italic>)-OHC beads but did not affect the binding of a fluorescent cyclopamine derivative (bodipy-cyclopamine) to Smo-expressing cells, showing that they engaged the CRD site but not the 7TM site (<xref ref-type="fig" rid="fig7">Figure 7B,C</xref>). The alkyne group was an important structural feature required for competition, as both 20(<italic>R</italic>)-OHC and 20-keto-cholesterol (<xref ref-type="fig" rid="fig7">Figure 7A</xref>) failed to inhibit the CRD–20(<italic>S</italic>)-OHC interaction (<xref ref-type="fig" rid="fig7">Figure 7B</xref>).<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.01340.016</object-id><label>Figure 7.</label><caption><title>Partial agonists that target the Smo CRD.</title><p>(<bold>A</bold>) Structure and synthetic logic for 20(<italic>R</italic>)-yne and 20-keto-yne. 20(<italic>R</italic>)-OHC and 20-keto-cholesterol are related analogs that lack the alkyne moiety. (<bold>B</bold>) Immunoblots show the amount of mSmo CRD-Fc captured on 20(<italic>S</italic>)-OHC beads in the presence of the indicated oxysterols added as competitors. (<bold>C</bold>) Binding of bodipy-cyclopamine to cells expressing full-length mSmo was determined by FACS in the presence of various Smo ligands. The bodipy-cyclopamine fluorescence in a cell population is expressed as a cumulative distribution function (CDF), which denotes the percentage of cells that show a given level of fluorescence or lower. Bodipy-cyclopamine binding can be competed by SANT-1 and Vismodegib, two 7TM site ligands, but not by any of the CRD-binding oxysterols. (<bold>D</bold>) Hh reporter activity in cells treated with increasing concentrations of the indicated oxysterols.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.016">http://dx.doi.org/10.7554/eLife.01340.016</ext-link></p></caption><graphic xlink:href="elife01340f007"/></fig></p><p>Despite binding to the mSmo CRD, 20(<italic>R</italic>)-yne and 20-keto-yne were weak activators of signaling in the absence of Shh, reinforcing the importance of stereochemistry at position 20 for Smo activation (<xref ref-type="fig" rid="fig7">Figure 7D</xref>). However, both molecules inhibited signaling induced by the native ligand Shh, the CRD agonist 20(<italic>S</italic>)-OHC or the 7TM agonist SAG (<xref ref-type="fig" rid="fig8">Figure 8A–C</xref> and <xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1</xref>). Both the molecules also reduced signaling by mSmoM2, a constitutively active, oncogenic Smo mutant (<xref ref-type="bibr" rid="bib78">Taipale et al., 2000</xref>), and mSmo D477H, the mouse version of a human Smo mutant that is resistant to the FDA-approved drug Vismodegib (<xref ref-type="fig" rid="fig8">Figure 8D,E</xref>) (<xref ref-type="bibr" rid="bib91">Yauch et al., 2009</xref>). We hereafter call these molecules oxysterol-based inhibitors or OBIs. This activity profile shows that the OBIs are CRD-targeted partial agonists of Smo that can reduce signaling by Smo activators and by clinically relevant Smo mutants. Our OBIs seem to inhibit Smo by a different mechanism compared to another recently reported CRD antagonist, 22-azacholesterol, which does not block signaling induced by SAG or by mSmoM2 (<xref ref-type="bibr" rid="bib55">Nedelcu et al., 2013</xref>). The broader Hh inhibitory activity of OBIs is instead reminiscent of the glucocorticoids Budesonide and Ciclesonide, which also fail to compete with cyclopamine for binding to Smo (<xref ref-type="bibr" rid="bib87">Wang et al., 2012</xref>).<fig-group><fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.01340.017</object-id><label>Figure 8.</label><caption><title>20(<italic>R</italic>)-yne and 20-keto-yne can inhibit Hh signaling.</title><p>(<bold>A–C</bold>) Hh reporter activation by 100 nM SAG (<bold>A</bold>), 5 μM 20(<italic>S</italic>)-OHC (<bold>B</bold>) and Shh (<bold>C</bold>) can be inhibited by both 20(<italic>R</italic>)-yne and 20-keto-yne. (<bold>D</bold>) Hh reporter activity in Smo<sup>−/−</sup> cells transfected with wild-type mSmo or mSmo D477H and then treated (48 hr) with Shh in the presence of 20(<italic>R</italic>)-yne, 20-keto-yne (both at 25 µM) or vismodegib (100 nM). (<bold>E</bold>) Hh reporter activity in NIH 3T3 cells transfected with constitutively active, oncogenic mSmoM2 and then treated (12 hr) with 20(<italic>R</italic>)-yne and 20-keto-yne (10 µM each) or cyclopamine (1 µM). (<bold>F</bold>) Accumulation of endogenous mSmo in cilia of NIH 3T3 cells treated (4 hr) with the indicated Smo ligands in the presence or absence of Shh. Each point represents the Smo fluorescence in a single cilium and the red lines denote the median and the interquartile range of mSmo fluorescence (n = 60 for each condition). (<bold>G</bold>) The binding of mSmo CRD-Fc to 20(<italic>S</italic>)-OHC beads can be inhibited by cyclopamine but not by the structurally-related alkaloid tomatidine. Error bars denote S.D. (n = 3).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.017">http://dx.doi.org/10.7554/eLife.01340.017</ext-link></p></caption><graphic xlink:href="elife01340f008"/></fig><fig id="fig8s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01340.018</object-id><label>Figure 8—figure supplement 1.</label><caption><title>Table of IC50 values for the OBIs.</title><p>In <xref ref-type="fig" rid="fig8">Figure 8A,B,C</xref>, we demonstrate the inhibitory activity of 20(<italic>R</italic>)-yne and 20-keto-yne in the presence of three different Hh activators (SAG, 20(<italic>S</italic>)-OHC and Shh). Using curve fitting (described in detail in methods), we derived IC50 values for each inhibitor in the presence of the three different activators. Values represent mean ± SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01340.018">http://dx.doi.org/10.7554/eLife.01340.018</ext-link></p></caption><graphic xlink:href="elife01340fs007"/></fig></fig-group></p><p>An early step in signaling that precedes transcription is the Shh-induced accumulation of Smo in the primary cilium (<xref ref-type="bibr" rid="bib16">Corbit et al., 2005</xref>). Antagonists that bind to the 7TM site display striking differences in their impact on this key trafficking step. Cyclopamine and cyclopamine derivatives (that contain a sterol-like tetracyclic ring structure) do not block Smo ciliary accumulation (<xref ref-type="fig" rid="fig8">Figure 8F</xref>) and in fact can drive Smo accumulation in cilia even in the absence of Shh (<xref ref-type="bibr" rid="bib64">Rohatgi et al., 2009</xref>). On the other hand, non-sterol 7TM antagonists like SANT-1 and Vismodegib prevent Shh-induced Smo accumulation in cilia (<xref ref-type="bibr" rid="bib64">Rohatgi et al., 2009</xref>). The CRD-targeted OBIs both behaved like cyclopamine in this assay—they induced Smo accumulation in cilia when added alone and also did not block Shh-induced ciliary accumulation of Smo (<xref ref-type="fig" rid="fig8">Figure 8F</xref>). This similarity between the OBIs and cyclopamine led us to consider the possibility that cyclopamine might not be a pure 7TM inhibitor like SANT-1 and Vismodegib but instead may also engage the CRD. Indeed, unlike the non-sterol 7TM inhibitors (<xref ref-type="fig" rid="fig2">Figure 2E</xref>), cyclopamine blocked the interaction between the mSmo CRD-Fc and 20(<italic>S</italic>)-OHC beads (<xref ref-type="fig" rid="fig8">Figure 8G</xref>), suggesting that it is capable of binding the CRD in this in vitro assay.</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>Our work provides both structural and mechanistic insights into the enigmatic CRD of Smo in Hh signaling. The CRD of Fz proteins binds to Wnt ligands. While the Fz CRD is related to the Smo CRD, no protein ligand has been identified to date that directly binds to the Smo CRD, and its role in Smo function has not been defined. In <italic>Drosophila</italic>, deletion of the Smo ectodomain or the mutation of specific cysteine residues in the CRD completely inactivates the protein (<xref ref-type="bibr" rid="bib54">Nakano et al., 2004</xref>). In contrast, in cultured mouse cells, ΔCRD-Smo has basal activity in overexpression experiments (<xref ref-type="bibr" rid="bib51">Murone et al., 1999</xref>; <xref ref-type="bibr" rid="bib79">Taipale et al., 2002</xref>). In zebrafish embryos, ΔCRD-Smo can rescue phenotypes dependent on low-level signaling but not on high-level signaling, and it shows higher levels of basal accumulation in cilia (<xref ref-type="bibr" rid="bib1">Aanstad et al., 2009</xref>). Our work now shows that the Smo CRD in vertebrates binds to oxysterols and mediates the ability of these lipids to activate Hh signaling. Structure-guided mutagenesis studies revealed that the Smo CRD binds to 20(<italic>S</italic>)-OHC in the region that was previously identified as the binding site for small hydrophobic molecules in other CRDs, formed by the evolutionary conserved helical bundle of the CRD core. This supports the hypothesis that CRDs evolved from an ancestral domain that sensed hydrophobic molecules (<xref ref-type="bibr" rid="bib6">Bazan and de Sauvage, 2009</xref>). Our structural analysis showed that the Smo CRD oxysterol binding site is most similar to the palmitoleyl-binding site in Fz CRDs (<xref ref-type="bibr" rid="bib31">Janda et al., 2012</xref>); however, the binding grooves are built of divergent residues (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref> and <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>), suggesting that they accommodate different classes of hydrophobic ligands.</p><p>Smo activity can be regulated by two distinct binding sites in the CRD and the 7TM segments. Oxysterols and their derivatives regulate Smo through the CRD site, while SANT-1, SAG, and Vismodegib bind to the 7TM segment. Binding of agonists like 20(<italic>S</italic>)-OHC and 20(<italic>S</italic>)-yne to the CRD must be communicated to the 7TM helix bundle for transduction across the membrane. Indeed, while the 7TM and CRD sites are separable, the dramatic synergy between 20(<italic>S</italic>)-OHC and SAG we have previously reported suggests a positive allosteric link between these domains (<xref ref-type="bibr" rid="bib53">Nachtergaele et al., 2012</xref>). This synergy also implies that 7TM and CRD ligands can bind to Smo simultaneously.</p><p>A speculative but intriguing insight into the interaction between the CRD and 7TM domains comes from our unexpected finding that the Hh inhibitor cyclopamine, an established 7TM ligand, also inhibits the binding of the isolated CRD to 20(<italic>S</italic>)-OHC beads. This result was unexpected because oxysterols do not decrease the binding of bodipy-cyclopamine to cells expressing full-length Smo (<xref ref-type="fig" rid="fig7">Figure 7C</xref> and <xref ref-type="bibr" rid="bib24">Dwyer et al., 2007</xref>). We believe that this discrepancy is due to the fact that the cell-based bodipy-cyclopamine binding assay is mostly measuring the interaction between cyclopamine and its high affinity (K<sub>d</sub>∼10 nM, <xref ref-type="bibr" rid="bib66">Rominger et al., 2009</xref>) binding site in the 7TM domain. Cell (or membrane) binding assays can often miss lower affinity (∼1–10 μM) interactions, which can be detected by ligand affinity chromatography assays (<xref ref-type="bibr" rid="bib59">Phizicky and Fields, 1995</xref>). It is also possible that cyclopamine binds the CRD more weakly when the CRD is embedded in the context of the whole protein.</p><p>As noted above, cyclopamine is a sterol that induces the accumulation of Smo in primary cilia, both properties that distinguish it from the pure 7TM site inhibitors SANT-1 and Vismodegib. One possibility is that cyclopamine can bridge the two ligand-binding sites on Smo and engage both a high-affinity interaction with the 7TM segment and a lower affinity interaction with the CRD. Alternatively, two molecules of cyclopamine could engage the CRD and 7TM sites separately or cyclopamine could be involved in a ‘hand-off’ interaction between the CRD and the 7TM segments analogous to the manner in which cholesterol is transferred between NPC2 and NPC1 (<xref ref-type="bibr" rid="bib38">Kwon et al., 2009</xref>; <xref ref-type="bibr" rid="bib86">Wang et al., 2010</xref>). While the relevance of this interaction for the inhibition of Smo by cyclopamine in cells remains to be established, the puzzling ability of cyclopamine to induce Smo accumulation in cilia (while inhibiting Smo activity) may be related to its ability to engage the CRD. This represents a third mechanism by which ligands can engage Smo, one that is distinct from pure 7TM and CRD ligands. Interestingly, glucocorticoids have been shown to fall into two distinct classes of Smo modulators—cyclopamine-competitive ligands that presumably bind to the 7TM potentiate signaling and a second class of inhibitors that do not compete with cyclopamine but appear to engage a distinct site (<xref ref-type="bibr" rid="bib87">Wang et al., 2012</xref>).</p><p>The CRD of Smo is also important for signaling by Shh, since ΔCRD-Smo cannot be efficiently activated by either Shh or 20(<italic>S</italic>)-OHC but remains responsive to SAG. While we observed very little activation of ΔCRD-YFP-Smo by Shh (<xref ref-type="fig" rid="fig3">Figure 3B</xref>), another study (<xref ref-type="bibr" rid="bib55">Nedelcu et al., 2013</xref>) reported that a ΔCRD-Smo-mCherry protein retained a low level of Shh responsiveness, suggesting that the CRD is not absolutely required for signaling initiated by Shh. This difference in the degree of Shh-responsiveness may be due to the position of the fluorescent protein tag, differences in the tendency of the YFP and mCherry tags to oligomerize or differences in the expression systems used in the two studies.</p><p>The striking decrease in Shh-responsiveness when the CRD is deleted raises two questions—does Ptch1 regulate Smo through the oxysterol binding site in the CRD and is 20(<italic>S</italic>)-OHC an endogenous ligand for Smo? Our mutagenesis of the putative oxysterol binding site in the CRD sheds light on the first question. We find mutations in the mSmo CRD (Y134F and G115F, <xref ref-type="fig" rid="fig6">Figure 6F</xref>) that can dissociate the Shh and oxysterol responses. These mutants fail to bind or respond to 20(<italic>S</italic>)-OHC but can still respond to Shh. The simplest interpretation of these data is that the endogenous Smo ligand regulated by Ptch1 does not bind Smo in precisely the same site as 20(<italic>S</italic>)-OHC. In fact, we have previously reported (<xref ref-type="bibr" rid="bib53">Nachtergaele et al., 2012</xref>) that cyclopamine is much less potent against Shh-activated Smo compared to 20(<italic>S</italic>)-OHC-activated Smo, likely because the conformation adopted by Smo is different in response to these two agonists. Both of these findings suggest that 20(<italic>S</italic>)-OHC is not the Ptch1-regulated ligand that modulates Smo activity in response to Shh reception. It remains possible that a Shh-regulated ligand binds to the CRD in a manner that is distinct from that of 20(<italic>S</italic>)-OHC.</p><p>The CRD is required for Smo to adopt a fully active conformation in response to Shh (but it is dispensable when Smo is activated by the synthetic 7TM ligand SAG). In this view, the CRD would serve as a domain that allosterically activates the 7TM helix bundle in response to Shh. Some mutations (Smo Y134F, P168A) that abolish 20(<italic>S</italic>)-OHC responses do indeed substantially dampen the ability of Shh to activate Smo. The observation that CRD point mutations in Smo that block oxysterol binding also impair signaling by Hh ligands has been used to infer that oxysterol binding is required for physiological Smo signaling (<xref ref-type="bibr" rid="bib55">Nedelcu et al., 2013</xref>). While this hypothesis has substantial implications for Hh regulation in development and cancer, it remains to be determined if the CRD site in cells is occupied by oxysterols or by a different ligand, or if perturbations in endogenous oxysterol levels can modulate Hh signaling. Finally, testing the activity of oxysterol binding site mutants in the context of embryonic development or Hh-driven tumors is essential for elucidating the physiological function of this site and whether it plays a role in graded, low-level or high-level signaling.</p><p>We have developed partial agonists of Smo that bind to the CRD. Understanding the structural and mechanistic basis for this partial agonism is an important future goal. Remarkably, the simple inversion of the stereochemistry at C-20 converts a potent agonist into a weak partial agonist and an effective inhibitor of signaling. This stereochemical inversion presumably allows the molecule to trap Smo in a poorly active confirmation, likely one similar to that stabilized by cyclopamine, in which Smo is localized in cilia but is inactive. The structures of the OBIs suggest that Smo activation potential depends critically on the spatial orientation between the ring system and the <italic>iso</italic>-octyl chain of 20(<italic>S</italic>)-OHC. Regardless of the mechanism, inhibitors targeting the Smo CRD would provide an orthogonal approach to modulate Hh signaling in regeneration and cancer. Partial agonists offer the possibility of blocking unrestrained signaling (such as that seen in cancer) while preserving lower-level, physiological signaling (<xref ref-type="bibr" rid="bib61">Riese, 2011</xref>). This ability to attenuate Smo activity may be useful since currently used Smo antagonists cause significant side-effects, leading nearly half of the patients in some trials to discontinue treatment (<xref ref-type="bibr" rid="bib80">Tang et al., 2012</xref>).</p><p>Perhaps the most important question moving forward is to identify the Shh-regulated ligand that mediates the communication between Ptch1 and Smo and to understand how it regulates Smo through the 7TM and CRD sites. Structural studies of a Smo construct carrying both the 7TM segment and the CRD in complex with various ligands that engage either site or both sites will be essential to understand how Smo transmits the Hh signal across the membrane.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Cells and reagents</title><p>NIH 3T3 and 293T cells were obtained from ATCC (Manassas, VA), and 293F cells were obtained from Life Technologies (Grand Island, NY). The production of Smo<sup>−/−</sup>:YFP-mSmo, Smo<sup>−/−</sup>:ΔCRD-YFP-mSmo and Smo<sup>−/−</sup>:ΔC-YFP-mSmo stable lines is described below. SAG (<underline>></underline>95%) was from Enzo Life Sciences (Farmingdale, NY), cyclopamine (<underline>></underline>98%) from Toronto Research Chemicals (Toronto, Ontario, Canada), Itraconazole (<underline>></underline>98%) from Sigma (St. Louis, MO) and SANT-1 (<underline>></underline>95%) from EMD Millipore (Billerica, MA). All sterols except <italic>ent</italic>-20(<italic>S</italic>)-OHC, 20(<italic>S</italic>)-yne, 20(<italic>R</italic>)-OHC, 20(<italic>R</italic>)-yne, 20-keto-cholesterol, 20-keto-yne and 20(<italic>S</italic>)-amine were purchased from Steraloids (purity <underline>></underline>98%) (Newport, RI).</p></sec><sec id="s4-2"><title>Constructs</title><p>All mSmo mutants were made using Quickchange or PCR methods in the context of a previously described construct encoding full-length mouse Smo (UniProt P56726) with the coding sequence for yellow fluorescent protein (YFP) inserted immediately after the signal sequence (pCS2:YFP-mSmo; <xref ref-type="bibr" rid="bib64">Rohatgi et al., 2009</xref>). The ΔCRD-YFP-mSmo construct lacked amino acids (a.a.) 68–184, and the ΔC-YFP-mSmo construct was truncated after a.a. 574. The construct encoding mSmo CRD-Fc was made by cloning the mSmo sequence encoding the CRD (a.a. 1–183) into a pCX vector carrying a C-terminal human Fc tag. Constructs for mammalian expression of the extracellular region of zebrafish Smo (UniProt Q90X26; zSmo ectodomain: a.a. 29–195), of a C-terminal, intracellular truncation of zSmo (UniProt Q90X26; zSmo ΔC: a.a. 29-624) or the CRD of <italic>Drosophila</italic> Smo (UniProt P91682; dSmo CRD: a.a. 32–204), fused C-terminally with either a hexa-histidine, mono Venus or 1D4 epitope-tag that can bind selectively the Rho 1D4 antibody (<xref ref-type="bibr" rid="bib48">Molday and MacKenzie, 1983</xref>), were cloned into the pHLsec vector (<xref ref-type="bibr" rid="bib3">Aricescu et al., 2006</xref>). A construct for bacterial expression of the extracellular region of zebrafish Smo (UniProt Q90X26; zSmo-ectodomain: a.a. 29–212), fused C-terminally with a with a hexa-histidine (His6) tag, was cloned into the pET22b vector.</p></sec><sec id="s4-3"><title>Stable cell lines</title><p>Stable cell lines expressing YFP-mSmo, ΔCRD-YFP-mSmo and ΔC-YFP-mSmo were made by infecting Smo<sup>−/−</sup> cells with a retrovirus carrying these constructs cloned into pMSCVpuro. Retrovirus was generated by transfecting the MSCV:YFP-mSmo constructs into Bosc23 cells. The virus-containing media were used to infect Smo<sup>−/−</sup> MEFs, and stable integrants were selected with puromycin and cloned by FACS.</p></sec><sec id="s4-4"><title>Chemical synthesis (general methods)</title><p>We have previously reported the chemical synthesis of <italic>ent</italic>-20(<italic>S</italic>)-OHC, 20(<italic>S</italic>)-yne, 20(<italic>R</italic>)-OHC and 20-keto-cholesterol (<xref ref-type="bibr" rid="bib53">Nachtergaele et al., 2012</xref>). Full synthetic procedures are provided below for <bold>20-keto-yne</bold>, <bold>20(<italic>R</italic>)-yne</bold>, and <bold>20(<italic>S</italic>)-amine</bold>. Melting points were determined on a Kofler micro hot stage and were uncorrected. NMR spectra were recorded in CDCl<sub>3</sub>, at 300 MHz (<sup>1</sup>H) or 75 MHz (<sup>13</sup>C). Chemical shifts (δ) were reported downfield from internal Me4Si (δ: 0.00). HR FAB-MS determinations were made with the use of JEOL MStation (JMS-700) Mass Spectrometer, matrix m-nitrobenzyl alcohol, with NaI as necessary, using mass spectrometry facilities located at the University of Missouri–St. Louis. HIRES-MS determinations were made with the use of Thermo Orbitrap Velos Mass Spectrometer, using the facilities located at Washington University in St. Louis. IR spectra were recorded as films on a NaCl plate or in KBr. Elemental analyses were carried out by M–H–W laboratories. Optical rotations were measured on a Perkin-Elmer polarimeter, Model 341. Chromatography was performed using flash chromatography grade silica gel (32–63 μm; Scientific Adsorbents, Atlanta, GA). Dichloromethane was distilled over CaH prior to application. Tetrahydrofuran was distilled over Na/benzophenone just prior to application. All other chemicals were used as purchased without further purification. Organic extracts were dried over anhydrous Na<sub>2</sub>SO<sub>4</sub>.</p></sec><sec id="s4-5"><title>20(<italic>S</italic>)-OHC bead synthesis</title><p><fig id="fig9" position="float"><graphic xlink:href="elife01340f009"/></fig></p><p>20(<italic>S</italic>)-amine was prepared as a 10 mM stock in 1:1 chloroform/methanol. For each coupling reaction, 250 μl (packed volume) of FastFlow 4 NHS-activated sepharose (GE Healthcare, San Francisco, CA) was washed extensively into DMSO. 300 μl of DMSO, 2.5 μl of the 10 mM 20(<italic>S</italic>)-amine stock and 1.5 μl of triethylamine were added to the washed resin, and the reaction was rotated for 4 hr at room temperature, protected from light. After coupling, the beads were spun down, the supernatant removed and 1 ml of 5% ethanolamine in DMSO was added to block the remaining free reactive sites (4 hr, room temperature, protected from light).</p></sec><sec id="s4-6"><title>Hedgehog reporter assays</title><p>For reporter assays in NIH 3T3 cells, a 10-cm plate of cells was transfected with 8 μg of a 4:1 wt/wt ratio of firefly luciferase reporter driven by an 8xGli-responsive promoter (<xref ref-type="bibr" rid="bib68">Sasaki et al., 1997</xref>) and a Renilla luciferase reporter driven by a constitutive TK promoter (Promega, Madison, WI). The next day, transfected cells were seeded into a 96-well plate, grown to confluence, and treated overnight with drugs diluted in media containing 0.5% fetal bovine serum (FBS). For reporter assays in Smo<sup>−/−</sup> cells, 25,000 cells per well were seeded in a 24-well plate 24 hr prior to transfection. The next day, after a media replacement step, each well was transfected with 1 ng Smo construct and 500 ng of the reporter mix described above, using Xtreme Gene HP (Roche, Mannheim, Germany). After overnight transfection, the media were once again changed to fresh media. Cells were grown to confluence and treated with drugs diluted in media with 0.5% FBS for 48 hr. Activity of both reporters was measured using the Dual-Luciferase Reporter kit (Promega) and read on a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT). The Gli luciferase to Renilla luciferase ratio is reported as ‘Hedgehog reporter activity’. Each experiment, which included three technical replicates, was repeated at least three times.</p></sec><sec id="s4-7"><title>Protein expression and purification of mSmo CRD</title><p>pCX-mSmo CRD-Fc was produced by secretion (96 hr) into the media of 293F suspension cells (Life Technologies, Grand Island, NY) transfected with an expression construct. The collected media were cleared by centrifugation (10 min, 1000×<italic>g</italic>, 4°C), adjusted to pH 8.5, filtered through a 0.22 μm PVDF membrane and applied to a 1 ml Protein A Hitrap column (GE Healthcare). mSmo CRD-Fc was eluted from the Protein A column with 100 mM citrate pH 3.5, immediately adjusted to pH 8.5 and then loaded on a Superose 6 (10/300, GE Healthcare) gel filtration column equilibrated in 20 mM Tris pH 8.5, 150 mM NaCl. Monodisperse protein that eluted as a sharp peak (<xref ref-type="fig" rid="fig2">Figure 2A</xref>) was collected and used for binding assays. The purified mSmo CRD could not be cleaved away from the Fc tag efficiently and thus was used in assays as the fusion. In addition, it could not be heated above 37°C prior to SDS-PAGE electrophoresis because it underwent irreversible aggregation.</p></sec><sec id="s4-8"><title>Expression and purification of the zSMO ectodomain and dSmo CRD from mammalian cells</title><p>The zSmo ectodomain and dSmo CRD were expressed by transient transfection in HEK-293T cells (using an automated procedure, <xref ref-type="bibr" rid="bib92">Zhao et al., 2011</xref>). 5 days post-transfection, the conditioned medium was dialyzed (for 48 hr at 4°C), and the ectodomain constructs of zSmo or dSmo were purified by either immobilized Rho 1D4 antibody affinity chromatography using CNBr-Activated Sepharose (GE Healthcare) as described previously (<xref ref-type="bibr" rid="bib48">Molday and MacKenzie, 1983</xref>) or IMAC using Talon beads (Clontech, Mountain View, CA). Proteins were concentrated and further purified by size-exclusion chromatography (Superdex 200 16/60 column; GE Healthcare) in buffer containing 10 mM HEPES, pH 7.5, 150 mM NaCl.</p></sec><sec id="s4-9"><title>Expression and purification of native and selenomethionine (SeMet)-substituted zSmo ectodomain from <italic>E. coli</italic></title><p>The zSmo ectodomain used for crystallization and oxysterol binding assays was expressed in <italic>E. coli</italic> Rosetta(DE3)pLysS cells (Novagen/EMD Millipore) as inclusion bodies and purified as follows (protocol adapted from <xref ref-type="bibr" rid="bib10">Brown et al. (2002)</xref>). After cell lysis, the inclusion body pellets were washed four times and then solubilized in 8 M urea, 50 mM Tris-HCl, pH 8, and 100 mM NaCl. The solubilized protein was then purified via IMAC (Ni-Sepharose FastFlow; GE Healthcare) under denaturing conditions. After IMAC purification the eluted protein was reduced with 10 mM DTT and added drop-wise to 1 l of rapidly-stirring refold buffer (3 M urea, 150 mM Tris pH 8.5, 200 mM <italic>L</italic>-arginine, 1.5 mM reduced glutathione [GSH], 0.15 mM oxidized glutathione [GSSG]), which was then further stirred gently overnight at room temperature. The solution was then dialysed into 25 mM Tris pH 8.5, 10 mM NaCl at 4°C, filtered, loaded onto a 5 ml HiTrap QFF column (GE Healthcare) and eluted with an NaCl gradient (from 10 mM to 1 M NaCl). The eluted protein was concentrated and further purified via size exclusion chromatography (Superdex 75 16/60 [GE Healthcare] in 10 mM HEPES pH 7.5, 150 mM NaCl).</p><p>SeMet-labeled zSmo ectodomain was produced in <italic>E. coli</italic> strain B834 (DE3) (Novagen/EMD Millipore). Cells were grown in 2 l cultures at 310 K for 4 hr and after induction with 300 μM isopropyl β-D-1-thiogalactopyranoside, the temperature was then lowered to 298 K. Following incubation for further 20 hr, the cells were harvested and the protein was purified as described for the unlabeled zSmo ectodomain.</p></sec><sec id="s4-10"><title>Immunoblotting</title><p>Cultured cells stably expressing YFP-mSmo, ΔCRD-YFP-mSmo, or ΔC-YFP-mSmo were scraped into ice-cold PBS containing SigmaFast Protease inhibitor cocktail (Sigma) and collected as a pellet by centrifugation (1000×<italic>g</italic>, 10 min, 4°C). Cells were lysed (1 hr, 4°C) by agitation in modified RIPA buffer (50 mM sodium-Tris pH 7.4, 150 mM sodium chloride, 2% NP-40, 0.5% deoxycholate, 0.1% sodium-dodecyl sulfate [SDS], 1 mM dithiothreitol and the SigmaFast Protease inhibitor cocktail). After clarification (20,000×<italic>g</italic>, 45 min, 4°C), the protein concentration of each lysate was measured using the bicinchoninic acid assay (BCA, Pierce/Thermo Scientific, Rockford, IL). Lysate aliquots containing equal amount of total protein were fractionated on an 8% SDS-PAGE gel and transferred to a nitrocellulose membrane for immunoblotting with anti-Gli1 antibody (#L42B10, 1:500; Cell Signaling, Denvers, MA), anti-GFP antibody (1:5000; Novus, Littleton, CO), anti-Gli3 antibody (AF3690 1:200; R&D, Minneapolis, MN) and anti-p38 antibody (ab31828, 1:2000; Abcam, Cambridge, MA). In <xref ref-type="fig" rid="fig1 fig2 fig3 fig4">Figures 1–4</xref>, vertical dashed black lines represent non-contiguous lanes from the same immunoblot juxtaposed for clarity.</p></sec><sec id="s4-11"><title>Ligand affinity chromatography</title><p>293T cells transfected with constructs encoding mSmo variants were lysed in hypotonic SEAT buffer (250 mM sucrose, 1 mM EDTA, 10 mM acetic acid, 10 mM triethanolamine and the SigmaFast EDTA-Free protease inhibitor cocktail). After the removal of nuclei by centrifugation (900×<italic>g</italic>, 5 min, 4°C), membranes were pelleted by ultracentrifugation (95,000×<italic>g</italic>, 30 min) and solubilized in a n-dodecyl-β-D-maltopyranoside (DDM) extraction buffer (50 mM Tris pH 7.4, 150 mM NaCl, 10% vol/vol glycerol, 0.5% wt/vol DDM and the SigmaFast EDTA-Free protease inhibitor cocktail) for 2 hr at 4°C, followed by removal of insoluble material by ultracentrifugation (100,000×<italic>g</italic>, 30 min). This DDM membrane extract was incubated with 20(<italic>S</italic>)-OHC beads for 12 hr at 4°C to allow binding to equilibrium. After extensive washing, proteins captured on the beads were eluted with reducing SDS sample buffer. The presence of YFP-mSmo in these eluates was determined by quantitative immunoblotting with an anti-YFP antibody (NB600-308, 1:5000; Novus) and infrared imaging (Li-Cor Odyssey).</p><p>For ligand affinity chromatography with purified mSmo CRD-Fc or zSmo ectodomain, protein was diluted in 20 mM Tris pH 8.5, 150 mM NaCl, 0.3% octyl-glucoside prior to addition of competitors and 20(<italic>S</italic>)-OHC beads. After binding was allowed to proceed for 12 hr at 4°C, the resin was washed and captured protein was eluted as described above. The presence of mSmo CRD-Fc was measured by an anti-human HRP-coupled antibody (1:20,000) or anti-human IR800-coupled antibody (1:10,000; for all quantitation, detected by LiCor Odyssey). The presence of zSmo ectodomain protein was measured by colloidal Coomassie staining (GelCode Blue, Pierce/Thermo Scientific).</p></sec><sec id="s4-12"><title>Immunofluorescence to detect ciliary Smoothened</title><p>Cells were fixed with cold 4% paraformaldehyde (10 min, room temperature [RT]), washed with phosphate buffered saline (PBS; 3 times, 5 min each), placed in blocking solution (PBS, 1% (vol/vol) normal donkey serum, 0.1% (vol/vol) Triton X-100, 10 mg/ml bovine serum albumin) for 30 min at RT and then stained with primary antibodies (overnight, 4°C): anti-acetylated tubulin (#T6793; Sigma) at 1:3000 (vol/vol) dilution and anti-Smo (<xref ref-type="bibr" rid="bib65">Rohatgi et al., 2007</xref>) at 1:500 (vol/vol) dilution in blocking solution. After washing (three times, 5 min in PBS + 0.1% Triton X-100), Alexa-coupled secondary antibodies (Jackson ImmunoResearch) were applied (1:500 [vol/vol] dilution, 1 hr, RT). Finally, stained cells were washed in PBS (three times, 5 min) and mounted onto glass slides with Prolong Gold mounting media with DAPI (Life Technologies).</p></sec><sec id="s4-13"><title>Microscopy and image analysis</title><p>The fixed cells were imaged with a Leica SP8 laser scanning confocal microscope, using a 63× oil objective (NA 1.40) and 1.3× zoom. For the quantitative analysis of Smo levels in cilia, all images used for comparisons were taken with identical gain, offset, and laser power settings on the microscope. Non-manipulated maximum projections of z-stacks were used for quantitation (Fiji). A mask, constructed by automatically applying a threshold to the acetylated tubulin image, was then applied to the corresponding anti-Smo image to measure Smo fluorescence at cilia. Local background correction was performed by moving the mask to measure fluorescence at a nearby region, and this value was subtracted from the ciliary Smo fluorescence.</p></sec><sec id="s4-14"><title>Data analysis</title><p>All statistical analysis and curve fitting were done in GraphPad Prism. For microscopy data, the Smo fluorescence for each cilium was individually plotted, generating a scatter plot that represents variability in the data. To compare Smo levels between different conditions, the median and interquartile range are provided (n = 60 for each condition).</p><p>For Hh reporter assays, each point is reported as the mean ± standard deviation (SD) derived from triplicates. Each result in the paper was repeated at least three times with similar outcomes. Relative luciferase activity was calculated by dividing Gli luciferase by Renilla luciferase luminescence. Fold-change in reporter activity was calculated by dividing each replicate by the mean reporter activity of the vehicle-treated control. Normalized (% of max) Hh reporter activity was calculated by setting the maximum value of a set to 100% and zero to 0% using the ‘normalize’ function of GraphPad Prism. In all graphs, dotted lines are straight connectors between points, and solid lines represent non-linear curve fits of the data (all done in GraphPad Prism). In <xref ref-type="fig" rid="fig2 fig4">Figures 2C and 4E</xref>, the curves were fit using the ‘log(inhibitor) vs response—variable slope’ function of GraphPad Prism. The model used for this function was Y = Bottom + (Top–Bottom)/(1+10^([LogIC50-X]*HillSlope), where ‘Y’ represents bound zSmo as a percentage of the maximum bound (with zero competitor), ‘Top’ and ‘Bottom’ represent the plateaus at the beginning and end of the curve, respectively, and ‘X’ represents the concentration of free competitor added to the binding reaction. In <xref ref-type="fig" rid="fig2 fig4">Figures 2B and 4D</xref>, the curve was fit using the ‘one site—total and nonspecific binding’ function. The equation used for this fit incorporates both specific binding (specific = Bmax*X/[X+Kd]) and non-specific binding (nonspecific = NS*X + Background). ‘X’ in this case represents the sterol immobilized on the resin. In <xref ref-type="fig" rid="fig8">Figure 8A,B,C</xref>, the same ‘log(inhibitor) vs response—variable slope’ function as above was used to asses the IC50s of the OBIs in a Hh reporter assay.</p></sec><sec id="s4-15"><title>Crystallization and data collection</title><p>Prior to crystallization, the zSmo ectodomain from bacterial expression was concentrated to 7 mg/ml. Crystallization trials, using 100 nl protein solution plus 100 nl reservoir solution in sitting drop vapor diffusion format, were set up in 96-well Greiner plates using a Cartesian Technologies robot (<xref ref-type="bibr" rid="bib84">Walter et al., 2005</xref>). Crystallization plates were maintained at 20.5°C in a TAP Homebase storage vault and imaged via a Veeco visualization system (<xref ref-type="bibr" rid="bib44">Mayo et al., 2005</xref>). zSmo ectodomain native and selenomethionine-substituted crystals were obtained out of mother liquor containing 100 mM HEPES pH7.0, PEG 6000 20%, 10 mM ZnCl<sub>2</sub>.</p><p>X-ray diffraction data were collected at 100 K and crystals were treated with 25% (vol/vol) glycerol in mother liquor for cryo protection. Data were collected at beamline I03 at the Diamond Light Source, UK (native zSmo ectodomain), and at beamline ID23-EH1 (selenomethionine-substituted zSmo ectodomain) at the European Synchrotron Radiation Facility (ESRF), France. X-ray data were processed and scaled with the HKL suite (<xref ref-type="bibr" rid="bib56">Otwinowski and Minor, 1997</xref>) and XIA2 (<xref ref-type="bibr" rid="bib27">Evans, 2006</xref>; <xref ref-type="bibr" rid="bib33">Kabsch, 2010</xref>; <xref ref-type="bibr" rid="bib89">Winter, 2010</xref>). Data collection statistics are shown in <xref ref-type="table" rid="tbl1">Table 1</xref>.</p></sec><sec id="s4-16"><title>Structure determination, refinement and analyses of zSmo ectodomain</title><p>The zSmo ectodomain crystal structure was determined by single anomalous dispersion (SAD) analysis. The positions of three selenium atoms were determined using SHELXD (<xref ref-type="bibr" rid="bib70">Schneider and Sheldrick, 2002</xref>). This solution was used as an input into the AutoSol module of the PHENIX suite (<xref ref-type="bibr" rid="bib2">Adams et al., 2002</xref>) for phase calculation and improvement. The resulting map was of high quality and allowed tracing of the whole polypeptide chain (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1A</xref>). An initial model was built automatically using RESOLVE (<xref ref-type="bibr" rid="bib81">Terwilliger, 2003</xref>) and completed manually using COOT (<xref ref-type="bibr" rid="bib25">Emsley and Cowtan, 2004</xref>). Iterative rounds of refinement in autoBUSTER (<xref ref-type="bibr" rid="bib8">Blanc et al., 2004</xref>), PHENIX (<xref ref-type="bibr" rid="bib2">Adams et al., 2002</xref>) and REFMAC (<xref ref-type="bibr" rid="bib52">Murshudov et al., 1997</xref>) applying non-crystallographic symmetry restraints as well as manual building in COOT (<xref ref-type="bibr" rid="bib25">Emsley and Cowtan, 2004</xref>) resulted in a well-defined model for zSmo ectodomain that included two molecules in the asymmetric unit both composed of residues 41–158 (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1B</xref>). The zSmo ectodomain N- and C-terminal regions could not be traced due to missing electron density and were not included in the final model. The native structure was solved by molecular replacement using PHASER (<xref ref-type="bibr" rid="bib45">McCoy et al., 2007</xref>) with the SeMet-labeled structure as a search model and refined as described above for the SeMet-labeled protein. Crystallographic and Ramachandran statistics are given in <xref ref-type="table" rid="tbl1">Table 1</xref>. Stereochemical properties were assessed by MolProbity (<xref ref-type="bibr" rid="bib20">Davis et al., 2007</xref>). Superposition of CRD structures and root mean square deviation (RMSD) values were calculated for equivalent Cα atoms using program SHP (<xref ref-type="bibr" rid="bib76">Stuart et al., 1979</xref>; <xref ref-type="bibr" rid="bib62">Riffel et al., 2002</xref>). The phylogenetic tree for CRDs (<xref ref-type="fig" rid="fig5">Figure 5B</xref>) was prepared with program PHYLIP (<xref ref-type="bibr" rid="bib28">Felsenstein, 1989</xref>) with the summed structural correlation data presented in <xref ref-type="fig" rid="fig5s3">Figure 5—figure supplement 3</xref> to construct a distance matrix. The program VOLUMES (RE Esnouf, unpublished) was used with a 1.4 Å radius probe to analyze the CRD binding grooves of zSmo and Fz8. The analysis of evolutionary conserved residues among the CRDs of the Smoothened family members was based on 80 amino acid sequences of vertebrate Smo CRDs and was mapped onto the zSmo CRD crystal structure using ProtSkin (<xref ref-type="bibr" rid="bib21">Deprez et al., 2005</xref>).</p></sec><sec id="s4-17"><title>Molecular docking and homology modeling</title><p>The refined atomic coordinates of the zSmo CRD crystal structure were kept rigid during the molecular docking. The guanidinium group of Arg139 forms a hydrogen bond with a carbonyl oxygen of Arg139 in the neighboring molecule and occludes the oxysterol-binding pocket. Thus, the mmt180 rotamer (<xref ref-type="bibr" rid="bib40">Lovell et al., 2000</xref>) of Arg139 was used during docking and pocket analysis. Atomic coordinates of 20(<italic>S</italic>)-OHC were downloaded from PubChem (compound ID 121935, <xref ref-type="bibr" rid="bib88">Wang et al., 2009</xref>) and kept flexible during docking in AutoDock 4.2.5.1 using the Lamarckian genetic algorithm and default parameters (<xref ref-type="bibr" rid="bib50">Morris et al., 2009</xref>). Estimated inhibition constant, K<sub>i</sub> (dissociation constant of the zSmo CRD-20(<italic>S</italic>)-OHC-complex), was calculated using formula K<sub>i</sub> = exp(ΔG/[R*T]), where ΔG is a free energy of binding in kcal/mol, R is the gas constant 1.987 cal K<sup>−1</sup> mol<sup>−1</sup>, and T = 298.15 K. The homology model of dSmo CRD (Ile82-Thr204, UniProtKB ID P91682) was built using program MODELLER 9.9 (<xref ref-type="bibr" rid="bib26">Eswar et al., 2008</xref>) with the zSmo CRD structure as a template. The amino acid sequence identity between the corresponding CRD regions is 42%.</p></sec><sec id="s4-18"><title>Multi angle light scattering (MALS)</title><p>MALS analysis of purified and glycosylated zebrafish Smo ectodomain (expressed from mammalian cells) was performed using an analytical Superdex S200 10/30 size exclusion chromatography column (GE Heathcare) eluted in 150 mM NaCl, 10 mM HEPES pH 7.5 (flow rate 0.5 ml/min) with static light scattering (DAWN HELEOS II, Wyatt Technology, Santa Barbara, CA), differential refractive index (Optilab rEX, Wyatt Technology) and Agilent 1200 UV (Agilent Technologies, Santa Clara, CA) detectors. Data were analyzed using the program ASTRA (Wyatt Technology).</p></sec><sec id="s4-19"><title>Zebrafish strain and husbandry</title><p>Adult fishes were maintained on a 14-hr light/10-hr dark cycle at 28°C in the AVA (Singapore) certified IMCB Zebrafish Facility. Zebrafish strain used was <italic>Tg(eng2a:eGFP)</italic><sup><italic>i233</italic></sup> (<xref ref-type="bibr" rid="bib43">Maurya et al., 2011</xref>).</p></sec><sec id="s4-20"><title>Zebrafish oxysterol treatment and in situ hybridization</title><p>The embryos of <italic>Tg(eng2a:eGFP)</italic><sup><italic>i233</italic></sup> were dechorinated using pronase (Roche) at one cell stage. The well-developing ones at the 50% epiboly stage were selected and grown in fish water containing 50 μM 20(<italic>S</italic>)-OHC or 40 μM cyclopamine. Control embryos were kept in water containing the same amount of ethanol, used as the vehicle for the drugs. Standard in situ hybridization (ISH) was performed with anti-Dig alkaline phosphatase and chromogenic substrate NBT/BCIP as previously described (<xref ref-type="bibr" rid="bib57">Oxtoby and Jowett, 1993</xref>). <italic>ptch2</italic> (formerly <italic>ptc1</italic>) RNA probe was prepared from template as previously described (<xref ref-type="bibr" rid="bib15">Concordet et al., 1996</xref>).</p></sec><sec id="s4-21"><title>Chemical synthesis (detailed methods and characterization)</title><sec id="s4-21-1"><title>4-Bromo-1-trimethylsilyl-1-butyne; (1)</title><p><fig id="fig10" position="float"><graphic xlink:href="elife01340f010"/></fig></p><p>Prepared according to a known literature procedure (<xref ref-type="bibr" rid="bib22">Dieter and Chen, 2006</xref>), CBr<sub>4</sub> (8.5 g, 25.6 mmol) was added to a solution of commercially available <bold>4-trimethylsilyl-3-butyn-1-ol</bold> (2.0 g, 14.06 mmol) in dry dichloromethane (DCM; 40 ml) at −30°C under N<sub>2</sub>. The mixture was stirred vigorously for 10 min, until the CBr<sub>4</sub> was completely dissolved, whereupon a solution of PPh<sub>3</sub> (5.53 g, 21.09 mmol) in dry DCM (12 ml) was added dropwise. The reaction mixture was stirred at −30°C for 2 hr, after which the temperature was raised to 0°C and was allowed to slowly warm to RT over the next 2 hr. Upon completion, the reaction mixture was filtered through a pad of silica and concentrated in vacuo. The residue was purified by column chromatography on silica gel (100% hexane elution), to yield compound <bold>1</bold> as a colorless liquid (2.16 g, 75%). Analytical data are as previously reported (<xref ref-type="bibr" rid="bib53">Nachtergaele et al., 2012</xref>).</p></sec><sec id="s4-21-2"><title>(3β, 17β)-17-[(1<italic>R</italic>,<italic>S</italic>)-1-hydroxypent-4-yn-1-yl]-3-methoxymethoxyandrost-5-ene; (2)</title><p><fig id="fig11" position="float"><graphic xlink:href="elife01340f011"/></fig></p><p>Magnesium metal turnings (0.21 g, 8.66 mmol) in anhydrous Et<sub>2</sub>O (15 ml) under N<sub>2</sub> were stirred in a two-necked flask equipped with a condenser. Compound <bold>1</bold> (1.77 g, 8.66 mmol) was added, followed by a few drops of 1,2-dibromoethane. The reaction mixture was warmed slightly to 30°C and stirred vigorously until cloudiness was observed (∼1–3 min). The reaction mixture was stirred an additional 30 min at RT, until the magnesium turnings were mostly consumed and the solution turned a murky yellow color. The flask was then cooled to 0°C, and a solution of <bold>(3β, 17β)-3-methoxymethoxyandrost-5-ene-17-carboxaldehyde</bold> (0.26 g, 0.75 mmol, <xref ref-type="bibr" rid="bib53">Nachtergaele et al., 2012</xref>) in anhydrous THF (9 ml) was added dropwise to the reaction. After 10 min, two new spots were formed on TLC, indicating formation of a diastereomeric mixture, and the reaction was quenched with NH<sub>4</sub>Cl<sub>(aq)</sub> (10 ml). The phases were separated, and the aqueous phase was extracted with Et<sub>2</sub>O (3 × 25 ml). The combined organic fractions were then washed with brine (1 × 25 ml), dried over Na<sub>2</sub>SO<sub>4</sub>, and concentrated in vacuo. The residue was quickly filtered through a small silica gel column (acetone–hexane, gradient elution). The product was concentrated, re-dissolved in anhydrous THF (15 ml) and cooled to 0°C. TBAF (0.96 ml, 1M in THF) was then added dropwise, and the reaction was allowed to stir for 10 min H<sub>2</sub>O (25 ml) was then added, and the reaction mixture was extracted with EtOAc (3 × 25 ml). The combined organic fractions were then dried over Na<sub>2</sub>SO<sub>4</sub>, and concentrated in vacuo. The compound was purified by column chromatography on silica gel (acetone–hexanes, gradient elution), to yield a diastereomeric mixture of compound <bold>2</bold> as a white solid, in 93% over two steps.</p><p><bold>Compound 2(<italic>R</italic>)</bold>: m.p. 135–137°C (EtOAc-hexanes); [α]<sub>D</sub><sup>20</sup> =—24.0 (<italic>c</italic> = 0.15, CHCl<sub>3</sub>); IR: 3391, 2932, 1436, 1148, 1105, 1036 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>) δ 5.29 (br s, 1H, H-6), 4.62 (s, 2H, OC<italic>H</italic><sub>2</sub>OCH<sub>3</sub>), 3.55-3.66 (br m, 1H, HOC<italic>H</italic>), 3.30-3.40 (m, 1H, H-3), 3.30 (s, 3H, OCH<sub>2</sub>OC<italic>H</italic><sub>3</sub>), 0.85-2.05 (m, 25H) 0.95 (s, 3H, H-19), 0.72 (s, 3H, H-18); <sup>13</sup>C NMR (CDCl<sub>3</sub>) δ 141.0, 121.8, 94.9, 77.1, 84.8, 73.7, 68.9, 56.6, 56.3, 55.4, 50.3, 42.6, 40.1, 39.8, 37.4, 37.0, 35.5, 32.1, 31.9, 29.1, 25.6, 24.8, 21.1, 19.6, 14.9, 12.6; HR-FAB MS [M+Na]<sup>+</sup> calculated for C<sub>26</sub>H<sub>40</sub>O<sub>3</sub>Na<sup>+</sup>: 423.2875, found 423.2878.</p><p><bold>Compound 2(<italic>S</italic>)</bold>: m.p. 111–113°C (EtOAc-hexanes); [α]<sub>D</sub><sup>20</sup> =—48.7 (<italic>c</italic> = 0.22 , CHCl<sub>3</sub>); IR: 3270, 2933, 1628, 1437, 1148, 1101, 1040 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>) δ 5.35 (br s, 1H, H-6), 4.68 (s, 2H, OC<italic>H</italic><sub>2</sub>OCH<sub>3</sub>), 3.67-3.75 (br m, 1H, HOC<italic>H</italic>), 3.34-3.47 (m, 1H, H-3), 3.36 (s, 3H, OCH<sub>2</sub>OC<italic>H</italic><sub>3</sub>), 0.85-2.40 (m, 25H) 1.00 (s, 3H, H-19), 0.71 (s, 3H, H-18); <sup>13</sup>C NMR (CDCl<sub>3</sub>) δ 140.9, 121.8, 94.9, 84.7, 77.1, 72.4, 68.7, 56.7, 56.5, 55.4, 50.3, 41.9, 39.7, 39.0, 37.4, 36.9, 35.5, 32.1, 31.7, 29.1, 25.3, 24.3, 21.0, 19.6, 15.1, 12.9; HR-FAB MS [M+Na]<sup>+</sup> calculated for C<sub>26</sub>H<sub>40</sub>O<sub>3</sub>Na<sup>+</sup>: 423.2875, found 423.2869.</p></sec><sec id="s4-21-3"><title>(3β, 17β)-17-(1-oxopent-4-yn-1-yl)-androst-5-en-3-ol; (20-keto-yne)</title><p><fig id="fig12" position="float"><graphic xlink:href="elife01340f012"/></fig></p><p>To a stirred solution of compound <bold>2</bold> (0.14 g, 0.34 mmol) in acetone (7 ml), freshly prepared Jones Reagent was added dropwise (∼0.2 ml; 30% CrO<sub>3</sub>·30% H<sub>2</sub>SO<sub>4</sub>·40% H<sub>2</sub>O); adding until the reaction solution turned from green to yellow. Monitoring by TLC showed the two diastereomer spots corresponding to <bold>2(<italic>R,S</italic>)</bold>, to be converted into one product spot. The reaction was subsequently quenched with isopropanol (3 ml), and the resulting blue-green solution was filtered through a pad of silica and then concentrated in vacuo. The resulting solid was then dissolved in MeOH:H<sub>2</sub>O (20 ml:10 drops), whereupon acetyl chloride (0.5 ml) was added dropwise over 15 min, and the reaction was stirred for 16 hr. Upon completion, the reaction flask was cooled to 0°C and was neutralized with sat. NaHCO<sub>3(aq)</sub> (∼10 ml). The reaction mixture was extracted with DCM (4 × 10 ml), and the organic fractions were combined, dried over Na<sub>2</sub>SO<sub>4</sub>, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (acetone–hexane, gradient elution), to yield compound <bold>2</bold> as a white solid in 97% over two steps.</p><p><bold>Compound 20-keto-yne</bold>: m.p. 148–150°C (EtOAc-hexanes); [α]<sub>D</sub><sup>20</sup> = +15.4 (<italic>c</italic> = 0.19, CHCl<sub>3</sub>); IR: 3294, 2928, 1704, 1435, 1382, 1097, 1047 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>) δ 5.32 (br s, 1H, H-6), 3.45-3.58 (m, 1H, H-3), 0.96-2.64 (m, 25H) 0.98 (s, 3H, H-19), 0.60 (s, 3H, H-18); <sup>13</sup>C NMR (CDCl<sub>3</sub>) δ 209.2, 140.9, 121.4, 83.7, 71.7, 68.7, 62.9, 57.0, 50.0, 44.5, 43.0, 42.3, 39.0, 37.4, 36.6, 31.9, 31.0, 31.7, 24.6, 23.0, 21.2, 19.5, 13.5, 13.0; HR-FAB MS [M+Na]<sup>+</sup> calculated for C<sub>24</sub>H<sub>34</sub>O<sub>2</sub>Na<sup>+</sup>: 377.2457, found 377.2462.</p></sec><sec id="s4-21-4"><title>(3β, 17β)-17-[(2<italic>R</italic>)-2-hydroxy-hex-4-yn-2-yl]-androst-5-en-3-ol; (20(<italic>R</italic>)-yne)</title><p><fig id="fig13" position="float"><graphic xlink:href="elife01340f013"/></fig></p><p>Following a reported protocol (<xref ref-type="bibr" rid="bib47">Mijares et al., 1967</xref>), <bold>20-keto-yne</bold> (78 mg, 0.22 mmol) was dissolved in anhydrous Et<sub>2</sub>O (10 ml) under N<sub>2</sub> and cooled to −25°C. Methylmagnesium bromide (3M in Et<sub>2</sub>O; 0.37 ml, 1.10 mmol) was added dropwise, and the reaction mixture was allowed to warm to RT for 2 hr. The reaction was then diluted with Et<sub>2</sub>O (10 ml) and quenched with NH<sub>4</sub>Cl<sub>(aq)</sub> (10 ml). The phases were separated, and the aqueous phase was extracted with Et<sub>2</sub>O (3 × 10 ml). The combined organic fractions were then washed with brine (1 × 15 ml), dried over Na<sub>2</sub>SO<sub>4</sub>, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (EtOAc–toluene, gradient elution), to yield the desired <bold>20(<italic>R</italic>)-yne</bold> as a white solid in 85% yield.</p><p><bold>Compound 20(<italic>R</italic>)-yne</bold>: m.p. 154–157°C (EtOAc-hexanes); [α]<sub>D</sub><sup>20</sup> =—58.2 (<italic>c</italic> = 0.18 , CHCl<sub>3</sub>); IR: 3307, 2928, 1645, 1433, 1381, 1054 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>) δ 5.35 (d, 1H, <sup>3</sup><italic>J</italic> = 5.6 Hz, H-6), 3.44-3.57 (m, 1H, H-3), 1.14 (s, 3H, H-21), 1.01 (s, 3H, H-19), 0.87 (s, 3H, H-18); <sup>13</sup>C NMR (CDCl<sub>3</sub>) δ 141.0, 121.7, 85.3, 75.5, 71.9, 68.6, 59.1, 57.0, 50.2, 43.1, 42.4, 41.1, 40.4, 37.4, 36.7, 31.9, 31.8, 31.5, 26.3, 24.0, 23.3, 21.1, 19.6, 13.9, 13.5; HR-FAB MS [M+Na]<sup>+</sup> calculated for C<sub>25</sub>H<sub>38</sub>O<sub>2</sub>Na<sup>+</sup>: 393.2770, found 393.2771.</p></sec><sec id="s4-21-5"><title>(3β, 17β)-17-[(2<italic>S</italic>)-2-hydroxypent-4-ene-2-yl]-androst-5-en-3-ol; (3)</title><p><fig id="fig14" position="float"><graphic xlink:href="elife01340f014"/></fig></p><p>Compound <bold>3</bold> was made using Barbier reaction conditions (<xref ref-type="bibr" rid="bib5">Barbier, 1899</xref>) (one-pot Gringard reaction) following the procedure of <xref ref-type="bibr" rid="bib72">Shaw (1966)</xref>. A portion of the magnesium turnings (2.92 g, 120.2 mmol) and a portion of the total allyl bromide (2.95 ml, 34.1 mmol) were stirred in a flame-dried 3-neck flask, fitted with a reflux condenser and a dropping funnel, under N<sub>2</sub>, containing in anhydrous Et<sub>2</sub>O (100 ml). The reaction mixture was warmed slightly (30°C), a few drops of 1,2-dibromoethane were added and the reaction mixture was stirred vigorously for 5 min, or until the solution appeared cloudy. The heat source was removed, and a solution of pregnenolone (3.0 g, 9.48 mmol) and allyl bromide (7.0 ml, 81.5 mmol) in Et<sub>2</sub>O:THF (1:2, vol/vol; 230 ml) was added to the dropping funnel. A quarter of the steroid mixture (∼60 ml) was slowly added over 7 min, at which time, another portion of magnesium turnings was added (0.23 g, 9.5 mmol). This sequence of steroid addition followed by magnesium addition was repeated thrice. The mixture was then refluxed for 2 hr at 40°C. Upon completion, the reaction mixture was diluted with Et<sub>2</sub>O (50 ml) and quenched slowly with NH<sub>4</sub>Cl<sub>(aq)</sub> (50 ml). The reaction mixture was filtered through cotton, to remove any remaining magnesium turnings, and into a separatory funnel, where upon additional H<sub>2</sub>O (100 ml) was added. The organic layer was then extracted with Et<sub>2</sub>O (3 × 1000 ml), dried over Na<sub>2</sub>SO<sub>4</sub>, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (EtOAc–hexanes, gradient elution), to yield compound <bold>3</bold> as a mixture of diastereomers (<italic>R:S</italic>; 1:9) as a white solid in 98% yield. The desired <bold>3(<italic>S</italic>)</bold> diastereomer was obtained through recrystallization from DCM:MeOH. Spectral data were as previously reported (<xref ref-type="bibr" rid="bib14">Colonna and Gros, 1973</xref>).</p></sec><sec id="s4-21-6"><title>(3β, 17β)-3-methoxymethoxy-17-[(2<italic>S</italic>)-2-methoxymethoxy-pent-4-en-2-yl]-androst-5-ene; (4)</title><p><fig id="fig15" position="float"><graphic xlink:href="elife01340f015"/></fig></p><p>Compound <bold>3</bold> (3.37 g, 9.40 mmol) was dissolved in dry DCM (150 ml) in a 2-neck flask fitted with a reflux condenser at 0°C. Diisopropyl ethylamine (13.0 ml, 75.19 mmol) and a catalytic amount of DMAP (57 mg) were next added to the flask. MOMCl (2.86 ml, 37.59 mmol) was then added dropwise, and after 10 min, the reaction mixture was removed from the ice bath and was refluxed at 50° for 24 hr. Upon completion, the reaction was quenched with H<sub>2</sub>O (25 ml). The reaction mixture was then washed sequentially with sat. NaHCO<sub>3(aq)</sub> (50 ml) and NaCl<sub>(aq)</sub> (50 ml), dried over Na<sub>2</sub>SO<sub>4</sub>, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (EtOAc–hexanes, gradient elution), to yield the desired compound <bold>4</bold> as a white solid in 95% yield.</p><p><bold>Compound 4</bold>: m.p. 87–88°C (MeOH-DCM); IR: 2935, 2891, 2846, 1639, 1464, 1439, 1371, 1147, 1106, 1037, 916 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>) δ 5.62-5.78 (m, 1H, C<italic>H</italic>=CH<sub>2</sub>), 5.30 (br d, 1H, <sup>3</sup><italic>J</italic> = 5.6 Hz, H-6), 4.93-5.45 (2 d, 2H, CH=C<italic>H</italic><sub>2</sub>) 4.71 (dd, 1H, <italic>J</italic> = 1.2 Hz, <sup>3</sup><italic>J</italic> = 9.8 Hz C(17)-COC<italic>H</italic><sub>2</sub>OCH<sub>3</sub>), 4.62 (s, 2H, C(3)-OC<italic>H</italic><sub>2</sub>OCH<sub>3</sub>), 4.61 (dd, 1H, <italic>J</italic> = 1.2 Hz, <sup>3</sup><italic>J</italic> = 9.8 Hz, C(17)-COC<italic>H</italic><sub>2</sub>OCH<sub>3</sub>), 3.30-3.42 (m, 1H, H-3), 3.31 (2 s, 6H, C(17)-COCH<sub>2</sub>OC<italic>H</italic><sub>3</sub>, C(3)-COCH<sub>2</sub>OC<italic>H</italic><sub>3</sub>), 1.28 (s, 3H, C(17)-CC<italic>H</italic><sub>3</sub>), 0.96 (s, 1H, H-19), 0.80 (s, 3H, H-18); <sup>13</sup>C NMR (CDCl<sub>3</sub>) δ 140.8, 135.1, 121.7, 117.2, 94.7, 90.7, 80.0, 77.0, 57.4, 56.9, 55.5, 55.1, 50.2, 45.4, 42.6, 40.4, 39.6, 37.3, 36.8, 31.9, 31.4, 29.0, 23.9, 23.0, 22.1, 21.0, 19.4, 13.9; Anal. Calculated. for C<sub>28</sub>H<sub>46</sub>O<sub>4</sub>: C, 75.29; H, 10.38. Observed: C, 75.19; H, 10.48.</p></sec><sec id="s4-21-7"><title>(4S)-[4-[(3β, 17β)-3-methoxymethoxyandrost-5-en-17-yl]-4-methoxymethoxy]-pentan-1-ol; (5)</title><p><fig id="fig16" position="float"><graphic xlink:href="elife01340f016"/></fig></p><p>Compound <bold>4</bold> (2.5 g, 5.6 mmol) was dissolved in THF (50 ml) under N<sub>2</sub>, and 9-BBN (56 ml, 0.5M in THF) was added dropwise. The reaction mixture was allowed to stir at RT for 16 hr, and upon reaction completion [3N] NaOH<sub>(aq)</sub> (8 ml) was carefully added, followed by the careful addition of 30% H<sub>2</sub>O<sub>2</sub> (8 ml), and the reaction was stirred for 1 hr. H<sub>2</sub>O was added (50 ml), and the reaction mixture was extracted with EtOAc (3 × 50 ml), dried over Na<sub>2</sub>SO<sub>4</sub>, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (EtOAc–hexanes, gradient elution), to yield the desired compound <bold>5</bold> as a white solid in 96% yield.</p><p><bold>Compound 5</bold>: m.p. 87–90°C (cold MeOH); IR: 3436, 2934, 2899, 2848, 1464, 1439, 1370, 1147, 1105, 1036, 916 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>) δ 5.32 (br d, 1H, <sup>3</sup><italic>J</italic> = 6.8 Hz, H-6), 4.70 (d, 1H, <sup>3</sup><italic>J</italic> = 9.6 Hz, C(17)-COC<italic>H</italic><sub>2</sub>OCH<sub>3</sub>), 4.65 (s, 2H, C(3)-OC<italic>H</italic><sub>2</sub>OCH<sub>3</sub>), 4.63 (d, 1H, <sup>3</sup><italic>J</italic> = 9.6 Hz, C(17)-COC<italic>H</italic><sub>2</sub>OCH<sub>3</sub>), 3.52-3.61 (m, 2H, C<italic>H</italic><sub>2</sub>OH), 3.32-3.44 (m, 1H, H-3), 3.33 (2 s, 6H, C(17)-COCH<sub>2</sub>OC<italic>H</italic><sub>3</sub>, C(3)-COCH<sub>2</sub>OC<italic>H</italic><sub>3</sub>), 1.27 (s, 3H, C(17)-CC<italic>H</italic><sub>3</sub>), 0.98 (s, 1H, H-19), 0.80 (s, 3H, H-18); <sup>13</sup>C NMR (CDCl<sub>3</sub>) δ 140.8, 121.8, 94.8, 90.6, 80.6, 77.1, 63.3, 57.3, 57.1, 55.6, 55.3, 50.2, 42.6, 40.4, 39.7, 37.4, 36.8, 36.3, 31.9, 31.5, 29.0, 28.2, 23.9, 23.0, 22.7, 21.0, 19.4, 14.1; Anal. calculated for C<sub>28</sub>H<sub>48</sub>O<sub>5</sub>: C, 72.37; H, 10.41. Observed: C, 72.60; H, 10.16.</p></sec><sec id="s4-21-8"><title>(3β, 17β)-3-methoxymethoxy-17-[(2<italic>S</italic>)-5-azido-2-methoxymethoxy-pentan-2-yl]-androst-5-ene; (6)</title><p><fig id="fig17" position="float"><graphic xlink:href="elife01340f017"/></fig></p><p>In a similar manner to reported protocol (<xref ref-type="bibr" rid="bib60">Pore et al., 2006</xref>), compound <bold>5</bold> (1.51 g, 3.25 mmol) was dissolved in dry DCM (20 ml) under N<sub>2</sub>. The reaction mixture was cooled to 0°C, and Et<sub>3</sub>N (0.91 ml, 6.5 mmol) was added. A solution of methanesulfonyl chloride (0.38 ml, 4.87 mmol) in dry DCM (7 ml) was added dropwise to the reaction over 10 min. The reaction was allowed to stir for 30 min at 0°C. Upon reaction completion, ice water (10 ml) was added. The organic phase was separated and sequentially washed with sat. NaHCO<sub>3(aq)</sub> (10 ml), brine (10 ml), dried over Na<sub>2</sub>SO<sub>4</sub>, and concentrated in vacuo; taking care not to warm the flask over 30°C in the process. The crude mesylated steroid was allowed to dry under vacuum for 2–3 hr, upon which time it was transferred to a 2-neck flask fitted with a reflux condenser, and was re-dissolved in anhydrous DMF (50 ml). NaN<sub>3</sub> (1.06 g, 16.25 mmol) was then added, and the reaction was stirred at 65°C for 1 hr. The reaction was then dumped into a beaker of ice water (50 ml) to quench the NaN<sub>3</sub>, transferred to a separatory funnel and extracted with EtOAc:Et<sub>2</sub>O (1:2, vol/vol; 4 × 25 ml). The organic fractions were combined and dried over Na<sub>2</sub>SO<sub>4</sub> and concentrated in vacuo. The residue was purified by column chromatography on silica gel (EtOAc–hexanes, gradient elution), to yield the desired compound <bold>6</bold> as a white solid in 94% yield.</p><p><bold>Compound 6</bold>: m.p. 78–79°C (EtOAc:hexanes); IR: 2963, 2934, 2895, 2833, 2094, 1462, 1437, 1371, 1265, 1140, 1106, 1090, 1041, 914 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>) δ 5.33 (br d, 1H, <sup>3</sup><italic>J</italic> = 5.6 Hz, H-6), 4.73 (dd, 1H, <italic>J</italic> = 1.6 Hz, <sup>3</sup><italic>J</italic> = 9.4 Hz, C(17)-COC<italic>H</italic><sub>2</sub>OCH<sub>3</sub>), 4.66 (d, 2H, <italic>J</italic> = 1.6 Hz, C(3)-OC<italic>H</italic><sub>2</sub>OCH<sub>3</sub>), 4.62 (dd, 1H, <italic>J</italic> = 1.6 Hz, <sup>3</sup><italic>J</italic> = 9.4 Hz, C(17)-COC<italic>H</italic><sub>2</sub>OCH<sub>3</sub>), 3.34-3.46 (m, 1H, H-3), 3.34 (2 s, 6H, C(17)-COCH<sub>2</sub>OC<italic>H</italic><sub>3</sub>, C(3)-COCH<sub>2</sub>OC<italic>H</italic><sub>3</sub>), 3.18-3.27 (m, 2H, C<italic>H</italic><sub>2</sub>N<sub>3</sub>), 1.29 (s, 3H, C(17)-CC<italic>H</italic><sub>3</sub>), 0.99 (s, 1H, H-19), 0.81 (s, 3H, H-18); <sup>13</sup>C NMR (CDCl<sub>3</sub>) δ 140.9, 121.8, 94.8, 90.7, 80.3, 77.1, 57.4, 57.1, 55.6, 55.3, 52.2, 50.3, 42.7, 40.4, 39.7, 37.4 (x2), 36.9, 32.0, 31.5, 29.1, 24.6, 23.9, 23.0, 22.7, 21.0, 19.5, 14.1; Anal. calculated for C<sub>28</sub>H<sub>47</sub>N<sub>3</sub>O<sub>4</sub>: C, 68.68; H, 9.67; N, 8.58. Observed: C, 68.47; H, 9.48; N, 8.59.</p></sec><sec id="s4-21-9"><title>(3β, 17β)-17-[(2<italic>S</italic>)-5-azido-2-hydroxy-pentan-2-yl]-androst-5-en-3-ol; (7)</title><p><fig id="fig18" position="float"><graphic xlink:href="elife01340f018"/></fig></p><p>Compound <bold>6</bold> (750 mg, 1.53 mmol) was dissolved in MeOH:DCM (80 ml:20 ml) and cooled to 0°C. AcCl (2.5 ml) was added dropwise, and the reaction was kept between 0°C and 10°C to minimize competing elimination byproducts. After 2 days, the starting material was found to be completely consumed, and the reaction mixture was cooled down to 0°C, and quenched very slowly with sat. NaHCO<sub>3(aq)</sub>. The reaction was then diluted further with H<sub>2</sub>O and extracted with DCM (4 × 25 ml). The combined organic fractions were then dried over Na<sub>2</sub>SO<sub>4</sub> and concentrated in vacuo. The residue was purified by column chromatography on silica gel (EtOAc–hexanes, gradient elution), to yield the desired compound <bold>7</bold> as a white solid in 65% yield.</p><p><bold>Compound 7</bold>: m.p. 106–108°C (EtOAc:hexanes); [α]<sub>D</sub><sup>20</sup> =—52.87 (<italic>c</italic> = 0.23 , CHCl<sub>3</sub>); IR: 3400, 2935, 2902, 2868, 2097, 1464, 1376, 1353, 1260, 1055 cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>) δ 5.28 (br d, 1H, <sup>3</sup><italic>J</italic> = 5.6 Hz, H-6), 3.36-3.52 (m, 1H, H-3), 3.11-3.28 (m, 2H, C<italic>H</italic><sub>2</sub>N<sub>3</sub>), 1.21 (s, 3H, H-21), 0.93 (s, 3H, H-19), 0.79 (s, 3H, H-18); <sup>13</sup>C NMR (CDCl<sub>3</sub>) δ 141.0, 121.7, 75.1, 71.9, 58.4, 57.1, 52.2, 50.2, 42.9, 42.5, 40.6, 40.3, 37.4, 36.7, 31.9, 31.8, 31.5, 26.4, 24.0 (x2), 22.6, 21.1, 19.6, 13.7; Anal. Calculated for C<sub>24</sub>H<sub>39</sub>N<sub>3</sub>O<sub>2</sub>: C, 71.78; H, 9.79; N, 10.46. Observed: C, 71.68; H, 9.66; N, 10.27.</p></sec><sec id="s4-21-10"><title>(3β, 17β)-17-[(2<italic>S</italic>)-5-amino-2-hydroxy-pentan-2-yl]-androst-5-en-3-ol; (20(<italic>S</italic>)-amine)</title><p><fig id="fig19" position="float"><graphic xlink:href="elife01340f019"/></fig></p><p>In a similar manner to reported protocols (<xref ref-type="bibr" rid="bib93">Zhao and Zhong, 2005</xref>; <xref ref-type="bibr" rid="bib67">Ryu et al., 2006</xref>), compound <bold>7</bold> (150 mg, 0.374 mmol) was dissolved in THF:H<sub>2</sub>O (2.0 ml: 0.05 ml) and warmed to 30°C for 30 min. The reaction was then allowed to cool to room temperature and stirred for an additional 24 hr. The reaction was monitored by TLC (MeOH:EtOAC:Et<sub>3</sub>N, 1:1:0.1), and upon completion, the solvents were evaporated, keeping water bath below 35°C. The residue was then redissolved in a minimal amount of MeOH:DCM and purified by column chromatography on a silica gel column packed with hexanes:Et<sub>3</sub>N (49:1). Gradient elution was started using EtOAc-hexanes (adding ∼0.5% Et<sub>3</sub>N to each gradient) to remove PPh<sub>3</sub> byproducts; then transitioning to 100% DCM (still adding 0.5% Et<sub>3</sub>N); and the compound was finally eluted by flushing with 1% MeOH-DCM (still adding 0.5% Et<sub>3</sub>N) to give compound <bold>20(<italic>S</italic>)-amine</bold> in 90% yield as a white solid, without the need for further purification. Attempts to recrystallize the product from MeOH were unsuccessful, and prolonged periods in the solvent seemed induce the formation of a new spot on TLC, presumably an ammonium salt. Thus, the compound was best purified only by column chromatography.</p><p><bold>Compound 20(<italic>S</italic>)-amine</bold>: m.p. 167–172°C (amorphous solid; MeOH:DCM); IR: 3368, 2931, 2900, cm<sup>−1</sup>; <sup>1</sup>H NMR (CDCl<sub>3</sub>:MeOD; ∼20:1) δ 5.35 (d, 1H, <sup>3</sup><italic>J</italic> = 7.2 Hz, 1H, H-6), 3.46-3.59 (m, 1H, H-3), 2.70-2.82 (m, 1H, C<italic>H</italic><sub>2</sub>NH<sub>2</sub>), 2.58-2.70 (m, 1H, C<italic>H</italic><sub>2</sub>NH<sub>2</sub>), 2.17-2.35 (m, 2H), 2.06-2.15 (dt, 1H), 1.93-2.04 (m, 1H), 1.28 (s, 3H, H-21), 1.02 (s, 3H, H-19), 0.87 (s, 3H, H-18); <sup>13</sup>C NMR (CDCl<sub>3</sub>:MeOD; ∼20:1) δ 141.0, 121.4, 74.7, 71.3, 58.4, 57.0, 50.1, 42.6, 42.1, 42.0, 40.7, 40.2, 37.3, 36.5, 31.8, 31.3, 31.3, 27.4, 25.7, 23.8, 22.4, 20.9, 19.3, 13.4; HR-FAB MS [M+H]<sup>+</sup> calculated for C<sub>24</sub>H<sub>42</sub>NO<sub>2</sub><sup>+</sup>: 376.3216, found 376.3208.</p></sec></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank the staff of beamline I03 from the Diamond Light Source, UK, and beamline ID23-EH1 from the European Synchrotron Radiation Facility, France, for assistance with data collection, B Bishop for laboratory support, RM Esnouf for help with volume calculations, and Luigi De Colibus, Alex Evers, Pehr Harbury and Suzanne Pfeffer for helpful discussions. We thank C Hughes, G Pusapati, G Luchetti, and A Lebensohn for help with experiments and P Lovelace for help with FACS. Mass spectrometry analysis was conducted at the NIH/NCRR Mass Spectrometry Facility at Washington University, supported by the NIH (RR00954, DK020579, DK056341). The crystal structures presented in this work have been deposited under PDB codes 4C79 and 4C7A.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>SN, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con2"><p>DMW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con3"><p>LKM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con4"><p>TM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con5"><p>PWI, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con6"><p>DFC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con7"><p>CS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con8"><p>RR, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con9"><p>ZZ, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con10"><p>KK, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Animal experimentation: Zebrafish were maintained in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, inspected annually by Agri-Food and Veterinary Authority of Singapore and quarterly by Biological Resource Centre to ensure strict adherence to the stipulated animal welfare guidelines. All animals were handled according to approved institutional care and use committee (IUCAC) protocols (#110638 and #120751) of the National University of Singapore.</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><sec sec-type="datasets"><title>Major dataset</title><p>The following datasets were generated:</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro1"><name><surname>Nachtergaele</surname><given-names>S</given-names></name>, <name><surname>Whalen</surname><given-names>DM</given-names></name>, <name><surname>Mydock</surname><given-names>LK</given-names></name>, <name><surname>Zhao</surname><given-names>Z</given-names></name>, <name><surname>Malinauskas</surname><given-names>T</given-names></name>, <name><surname>Krishnan</surname><given-names>K</given-names></name>, <etal/>, <year>2013</year><x>, </x><source>Crystal structure of the Smoothened CRD, native</source><x>, </x><object-id pub-id-type="art-access-id">4C79</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4C79">http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4C79</ext-link><x>, </x><comment>Publicly available at the RCSB Protein Data Bank (<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/">http://www.rcsb.org/</ext-link>).</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro2"><name><surname>Nachtergaele</surname><given-names>S</given-names></name>, <name><surname>Whalen</surname><given-names>DM</given-names></name>, <name><surname>Mydock</surname><given-names>LK</given-names></name>, <name><surname>Zhao</surname><given-names>Z</given-names></name>, <name><surname>Malinauskas</surname><given-names>T</given-names></name>, <name><surname>Krishnan</surname><given-names>K</given-names></name>, <etal/>, <year>2013</year><x>, </x><source>Crystal structure of the Smoothened CRD, selenomethionine-labeled</source><x>, </x><object-id pub-id-type="art-access-id">4C7A</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4C7A">http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4C7A</ext-link><x>, </x><comment>Publicly available at the RCSB Protein Data Bank (<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/">http://www.rcsb.org/</ext-link>).</comment></related-object></p><p>The following previously published datasets were used:</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro3"><name><surname>Dann</surname><given-names>CE</given-names></name>, <name><surname>Hsieh</surname><given-names>JC</given-names></name>, <name><surname>Rattner</surname><given-names>A</given-names></name>, <name><surname>Sharma</surname><given-names>D</given-names></name>, <name><surname>Nathans</surname><given-names>J</given-names></name>, <name><surname>Leahy</surname><given-names>DJ</given-names></name>, <year>2001</year><x>, </x><source>Crystal structure of the cysteine-rich domain of secreted Frizzled-related protein 3 (SFRP-3;FZB)</source><x>, </x><object-id pub-id-type="art-access-id">1IJX</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=1ijx">http://www.rcsb.org/pdb/explore/explore.do?structureId=1ijx</ext-link><x>, </x><comment>Publicly available at the RCSB Protein Data Bank (<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/">http://www.rcsb.org/</ext-link>).</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro4"><name><surname>Janda</surname><given-names>CY</given-names></name>, <name><surname>Waghray</surname><given-names>D</given-names></name>, <name><surname>Levin</surname><given-names>AM</given-names></name>, <name><surname>Thomas</surname><given-names>C</given-names></name>, <name><surname>Garcia</surname><given-names>KC</given-names></name>, <year>2012</year><x>, </x><source>Crystal structure of XWnt8 in complex with the cysteine-rich domain of Frizzled 8</source><x>, </x><object-id pub-id-type="art-access-id">4F0A</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4f0a">http://www.rcsb.org/pdb/explore/explore.do?structureId=4f0a</ext-link><x>, </x><comment>Publicly available at the RCSB Protein Data Bank (<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/">http://www.rcsb.org/</ext-link>).</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro5"><name><surname>Stiegler</surname><given-names>AL</given-names></name>, <name><surname>Burden</surname><given-names>SJ</given-names></name>, <name><surname>Hubbard</surname><given-names>SR</given-names></name>, <year>2009</year><x>, </x><source>Crystal Structure of the Frizzled-like Cysteine-rich Domain of MuSK</source><x>, </x><object-id pub-id-type="art-access-id">3HKL</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=3hkl">http://www.rcsb.org/pdb/explore/explore.do?structureId=3hkl</ext-link><x>, </x><comment>Publicly available at the RCSB Protein Data Bank (<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/">http://www.rcsb.org/</ext-link>).</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro6"><name><surname>Kwon</surname><given-names>HJ</given-names></name>, <name><surname>Abi-Mosleh</surname><given-names>L</given-names></name>, <name><surname>Wang</surname><given-names>ML</given-names></name>, <name><surname>Deisenhofer</surname><given-names>J</given-names></name>, <name><surname>Goldstein</surname><given-names>JL</given-names></name>, <name><surname>Brown</surname><given-names>MS</given-names></name>, <etal/>, <year>2009</year><x>, </x><source>NPC1(NTD):cholesterol</source><x>, </x><object-id pub-id-type="art-access-id">3GKI</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=3gki">http://www.rcsb.org/pdb/explore/explore.do?structureId=3gki</ext-link><x>, </x><comment>Publicly available at the RCSB Protein Data Bank (<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/">http://www.rcsb.org/</ext-link>).</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro7"><name><surname>Monaco</surname><given-names>HL</given-names></name>, <year>1997</year><x>, </x><source>Crystal structure of the chicken riboflavin-binding protein. RFBP (this older dataset is not available in the RCSB Protein Databank)</source><comment>Available by email from Prof. Hugo L. Monaco (<ext-link ext-link-type="uri" xlink:href="http://monaco@sci.univr.it/">monaco@sci.univr.it</ext-link>), University of Verona, Italy.</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro8"><name><surname>Chen</surname><given-names>C</given-names></name>, <name><surname>Ke</surname><given-names>J</given-names></name>, <name><surname>Zhou</surname><given-names>XE</given-names></name>, <name><surname>Yi</surname><given-names>W</given-names></name>, <name><surname>Brunzelle</surname><given-names>JS</given-names></name>, <name><surname>Li</surname><given-names>J</given-names></name>, <etal/>, <year>2013</year><x>, </x><source>Crystal structure of human folate receptor alpha in complex with folic acid</source><x>, </x><object-id pub-id-type="art-access-id">4LRH</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4lrh">http://www.rcsb.org/pdb/explore/explore.do?structureId=4lrh</ext-link><x>, </x><comment>Publicly available at the RCSB Protein Data Bank (<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/">http://www.rcsb.org/</ext-link>).</comment></related-object></p></sec></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aanstad</surname><given-names>P</given-names></name><name><surname>Santos</surname><given-names>N</given-names></name><name><surname>Corbit</surname><given-names>KC</given-names></name><name><surname>Scherz</surname><given-names>PJ</given-names></name><name><surname>Trinh le</surname><given-names>A</given-names></name><name><surname>Salvenmoser</surname><given-names>W</given-names></name><etal/></person-group><year>2009</year><article-title>The extracellular domain of Smoothened regulates ciliary localization and is required for high-level Hh signaling</article-title><source>Curr Biol</source><volume>19</volume><fpage>1034</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2009.04.053</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Adams</surname><given-names>PD</given-names></name><name><surname>Grosse-Kunstleve</surname><given-names>RW</given-names></name><name><surname>Hung</surname><given-names>LW</given-names></name><name><surname>Loerger</surname><given-names>TR</given-names></name><name><surname>McCoy</surname><given-names>AJ</given-names></name><name><surname>Moriarty</surname><given-names>NW</given-names></name><etal/></person-group><year>2002</year><article-title>PHENIX: building new software for automated crystallographic structure determination</article-title><source>Acta Crystallogr D Biol Crystallogr</source><volume>58</volume><fpage>1948</fpage><lpage>54</lpage><pub-id pub-id-type="doi">10.1107/S0907444902016657</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aricescu</surname><given-names>AR</given-names></name><name><surname>Lu</surname><given-names>W</given-names></name><name><surname>Jones</surname><given-names>EY</given-names></name></person-group><year>2006</year><article-title>A time- and cost-efficient system for high-level protein production in mammalian cells</article-title><source>Acta Crystallogr D Biol Crystallogr</source><volume>62</volume><fpage>1243</fpage><lpage>50</lpage><pub-id pub-id-type="doi">10.1107/S0907444906029799</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bale</surname><given-names>AE</given-names></name></person-group><year>2002</year><article-title>Hedgehog signaling and human disease</article-title><source>Annu Rev Genomics Human Genet</source><volume>3</volume><fpage>47</fpage><lpage>65</lpage><pub-id pub-id-type="doi">10.1146/annurev.genom.3.022502.103031</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Barbier</surname><given-names>P</given-names></name></person-group><year>1899</year><article-title>Synthese du diethylheptenol</article-title><source>C R Hebd Seances Acad Sc</source><volume>128</volume><fpage>110</fpage></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bazan</surname><given-names>JF</given-names></name><name><surname>de Sauvage</surname><given-names>FJ</given-names></name></person-group><year>2009</year><article-title>Structural ties between cholesterol transport and morphogen signaling</article-title><source>Cell</source><volume>138</volume><fpage>1055</fpage><lpage>6</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2009.09.006</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bazan</surname><given-names>JF</given-names></name><name><surname>Janda</surname><given-names>CY</given-names></name><name><surname>Garcia</surname><given-names>KC</given-names></name></person-group><year>2012</year><article-title>Structural architecture and functional evolution of Wnts</article-title><source>Dev Cell</source><volume>23</volume><fpage>227</fpage><lpage>32</lpage><pub-id pub-id-type="doi">10.1016/j.devcel.2012.07.011</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Blanc</surname><given-names>E</given-names></name><name><surname>Roversi</surname><given-names>P</given-names></name><name><surname>Vonrhein</surname><given-names>C</given-names></name><name><surname>Flensburg</surname><given-names>C</given-names></name><name><surname>Lea</surname><given-names>SM</given-names></name><name><surname>Bricogne</surname><given-names>G</given-names></name></person-group><year>2004</year><article-title>Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT</article-title><source>Acta Crystallogr D Biol Crystallogr</source><volume>60</volume><fpage>2210</fpage><lpage>21</lpage><pub-id pub-id-type="doi">10.1107/S0907444904016427</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Briscoe</surname><given-names>J</given-names></name><name><surname>Therond</surname><given-names>PP</given-names></name></person-group><year>2013</year><article-title>The mechanisms of Hedgehog signalling and its roles in development and disease</article-title><source>Nat Rev Mol Cell Biol</source><volume>14</volume><fpage>418</fpage><lpage>31</lpage><pub-id pub-id-type="doi">10.1038/nrm3598</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brown</surname><given-names>J</given-names></name><name><surname>Esnouf</surname><given-names>RM</given-names></name><name><surname>Jones</surname><given-names>MA</given-names></name><name><surname>Linnell</surname><given-names>J</given-names></name><name><surname>Harlos</surname><given-names>K</given-names></name><name><surname>Hassan</surname><given-names>AB</given-names></name><etal/></person-group><year>2002</year><article-title>Structure of a functional IGF2R fragment determined from the anomalous scattering of sulfur</article-title><source>EMBO J</source><volume>21</volume><fpage>1054</fpage><lpage>62</lpage><pub-id pub-id-type="doi">10.1093/emboj/21.5.1054</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>C</given-names></name><name><surname>Ke</surname><given-names>J</given-names></name><name><surname>Zhou</surname><given-names>XE</given-names></name><name><surname>Yi</surname><given-names>W</given-names></name><name><surname>Brunzelle</surname><given-names>JS</given-names></name><name><surname>Li</surname><given-names>J</given-names></name><etal/></person-group><year>2013</year><article-title>Structural basis for molecular recognition of folic acid by folate receptors</article-title><source>Nature</source><volume>500</volume><fpage>486</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1038/nature12327</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>JK</given-names></name><name><surname>Taipale</surname><given-names>J</given-names></name><name><surname>Cooper</surname><given-names>MK</given-names></name><name><surname>Beachy</surname><given-names>PA</given-names></name></person-group><year>2002a</year><article-title>Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened</article-title><source>Genes Dev</source><volume>16</volume><fpage>2743</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1101/gad.1025302</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>JK</given-names></name><name><surname>Taipale</surname><given-names>J</given-names></name><name><surname>Young</surname><given-names>KE</given-names></name><name><surname>Maiti</surname><given-names>T</given-names></name><name><surname>Beachy</surname><given-names>PA</given-names></name></person-group><year>2002b</year><article-title>Small molecule modulation of Smoothened activity</article-title><source>Proc Natl Acad Sci USA</source><volume>99</volume><fpage>14071</fpage><lpage>6</lpage><pub-id pub-id-type="doi">10.1073/pnas.182542899</pub-id></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Colonna</surname><given-names>AO</given-names></name><name><surname>Gros</surname><given-names>EG</given-names></name></person-group><year>1973</year><article-title>Synthesis of 3 beta-Acetoxy-24-Norchola-5,20(22)-Dien-23-al - new Intermediate for an Improved synthesis of [24-C-14]-Cholesterol</article-title><source>J Steroid Biochem</source><volume>4</volume><fpage>171</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1016/0022-4731(73)90023-X</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Concordet</surname><given-names>JP</given-names></name><name><surname>Lewis</surname><given-names>KE</given-names></name><name><surname>Moore</surname><given-names>JW</given-names></name><name><surname>Goodrich</surname><given-names>LV</given-names></name><name><surname>Johnson</surname><given-names>RL</given-names></name><name><surname>Scott</surname><given-names>MP</given-names></name><etal/></person-group><year>1996</year><article-title>Spatial regulation of a zebrafish patched homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning</article-title><source>Development</source><volume>122</volume><fpage>2835</fpage><lpage>46</lpage></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Corbit</surname><given-names>KC</given-names></name><name><surname>Aanstad</surname><given-names>P</given-names></name><name><surname>Singla</surname><given-names>V</given-names></name><name><surname>Norman</surname><given-names>AR</given-names></name><name><surname>Stainier</surname><given-names>DY</given-names></name><name><surname>Reiter</surname><given-names>JF</given-names></name></person-group><year>2005</year><article-title>Vertebrate Smoothened functions at the primary cilium</article-title><source>Nature</source><volume>437</volume><fpage>1018</fpage><lpage>21</lpage><pub-id pub-id-type="doi">10.1038/nature04117</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Corcoran</surname><given-names>RB</given-names></name><name><surname>Scott</surname><given-names>MP</given-names></name></person-group><year>2006</year><article-title>Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells</article-title><source>Proc Natl Acad Sci USA</source><volume>103</volume><fpage>8408</fpage><lpage>13</lpage><pub-id pub-id-type="doi">10.1073/pnas.0602852103</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dann</surname><given-names>CE</given-names></name><name><surname>Hsieh</surname><given-names>JC</given-names></name><name><surname>Rattner</surname><given-names>A</given-names></name><name><surname>Sharma</surname><given-names>D</given-names></name><name><surname>Nathans</surname><given-names>J</given-names></name><name><surname>Leahy</surname><given-names>DJ</given-names></name></person-group><year>2001</year><article-title>Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains</article-title><source>Nature</source><volume>412</volume><fpage>86</fpage><lpage>90</lpage><pub-id pub-id-type="doi">10.1038/35083601</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Davies</surname><given-names>JP</given-names></name><name><surname>Chen</surname><given-names>FW</given-names></name><name><surname>Ioannou</surname><given-names>YA</given-names></name></person-group><year>2000</year><article-title>Transmembrane molecular pump activity of Niemann-Pick C1 protein</article-title><source>Science</source><volume>290</volume><fpage>2295</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1126/science.290.5500.2295</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Davis</surname><given-names>IW</given-names></name><name><surname>Leaver-Fay</surname><given-names>A</given-names></name><name><surname>Chen</surname><given-names>VB</given-names></name><name><surname>Block</surname><given-names>JN</given-names></name><name><surname>Kapral</surname><given-names>GJ</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name><etal/></person-group><year>2007</year><article-title>MolProbity: all-atom contacts and structure validation for proteins and nucleic acids</article-title><source>Nucleic Acids Res</source><volume>35</volume><fpage>W375</fpage><lpage>83</lpage><pub-id pub-id-type="doi">10.1093/nar/gkm216</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Deprez</surname><given-names>C</given-names></name><name><surname>Lloubes</surname><given-names>R</given-names></name><name><surname>Gavioli</surname><given-names>M</given-names></name><name><surname>Marion</surname><given-names>D</given-names></name><name><surname>Guerlesquin</surname><given-names>F</given-names></name><name><surname>Blanchard</surname><given-names>L</given-names></name></person-group><year>2005</year><article-title>Solution structure of the <italic>E. coli</italic> TolA C-terminal domain reveals conformational changes upon binding to the phage g3p N-terminal domain</article-title><source>J Mol Biol</source><volume>346</volume><fpage>1047</fpage><lpage>57</lpage><pub-id pub-id-type="doi">10.1016/j.jmb.2004.12.028</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dieter</surname><given-names>RK</given-names></name><name><surname>Chen</surname><given-names>NY</given-names></name></person-group><year>2006</year><article-title>Asymmetric synthesis of enantioenriched (+)-elaeokanine A</article-title><source>J Org Chem</source><volume>71</volume><fpage>5674</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1021/jo060717q</pub-id></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dijkgraaf</surname><given-names>GJ</given-names></name><name><surname>Alicke</surname><given-names>B</given-names></name><name><surname>Weinmann</surname><given-names>L</given-names></name><name><surname>Januario</surname><given-names>T</given-names></name><name><surname>West</surname><given-names>K</given-names></name><name><surname>Modrusan</surname><given-names>Z</given-names></name><etal/></person-group><year>2011</year><article-title>Small molecule inhibition of GDC-0449 refractory smoothened mutants and downstream mechanisms of drug resistance</article-title><source>Cancer Res</source><volume>71</volume><fpage>435</fpage><lpage>44</lpage><pub-id pub-id-type="doi">10.1158/0008-5472.CAN-10-2876</pub-id></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dwyer</surname><given-names>JR</given-names></name><name><surname>Sever</surname><given-names>N</given-names></name><name><surname>Carlson</surname><given-names>M</given-names></name><name><surname>Nelson</surname><given-names>SF</given-names></name><name><surname>Beachy</surname><given-names>PA</given-names></name><name><surname>Parhami</surname><given-names>F</given-names></name></person-group><year>2007</year><article-title>Oxysterols are novel activators of the hedgehog signaling pathway in pluripotent mesenchymal cells</article-title><source>J Biol Chem</source><volume>282</volume><fpage>8959</fpage><lpage>68</lpage><pub-id pub-id-type="doi">10.1074/jbc.M611741200</pub-id></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Emsley</surname><given-names>P</given-names></name><name><surname>Cowtan</surname><given-names>K</given-names></name></person-group><year>2004</year><article-title>Coot: model-building tools for molecular graphics</article-title><source>Acta Crystallogr D Biol Crystallogr</source><volume>60</volume><fpage>2126</fpage><lpage>32</lpage><pub-id pub-id-type="doi">10.1107/S0907444904019158</pub-id></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Eswar</surname><given-names>N</given-names></name><name><surname>Eramian</surname><given-names>D</given-names></name><name><surname>Webb</surname><given-names>B</given-names></name><name><surname>Shen</surname><given-names>M</given-names></name><name><surname>Sali</surname><given-names>A</given-names></name></person-group><year>2008</year><article-title>Protein structure modeling with MODELLER</article-title><source>Methods Mol Biol</source><volume>426</volume><fpage>145</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1007/978-1-60327-058-8_8</pub-id></element-citation></ref><ref id="bib27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Evans</surname><given-names>P</given-names></name></person-group><year>2006</year><article-title>Scaling and assessment of data quality</article-title><source>Acta Crystallogr D Biol Crystallogr</source><volume>62</volume><fpage>72</fpage><lpage>82</lpage><pub-id pub-id-type="doi">10.1107/S0907444905036693</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Felsenstein</surname><given-names>J</given-names></name></person-group><year>1989</year><article-title>PHYLIP—Phylogeny Inference Package (Version 3.2)</article-title><source>Cladistics</source><volume>5</volume><fpage>164</fpage><lpage>6</lpage></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Frank-Kamenetsky</surname><given-names>M</given-names></name><name><surname>Zhang</surname><given-names>XM</given-names></name><name><surname>Bottega</surname><given-names>S</given-names></name><name><surname>Guicherit</surname><given-names>O</given-names></name><name><surname>Wichterle</surname><given-names>H</given-names></name><name><surname>Dudek</surname><given-names>H</given-names></name><etal/></person-group><year>2002</year><article-title>Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists</article-title><source>J Biol</source><volume>1</volume><fpage>10</fpage><pub-id pub-id-type="doi">10.1186/1475-4924-1-10</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fredriksson</surname><given-names>R</given-names></name><name><surname>Lagerstrom</surname><given-names>MC</given-names></name><name><surname>Lundin</surname><given-names>LG</given-names></name><name><surname>Schioth</surname><given-names>HB</given-names></name></person-group><year>2003</year><article-title>The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints</article-title><source>Mol Pharmacol</source><volume>63</volume><fpage>1256</fpage><lpage>72</lpage><pub-id pub-id-type="doi">10.1124/mol.63.6.1256</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Janda</surname><given-names>CY</given-names></name><name><surname>Waghray</surname><given-names>D</given-names></name><name><surname>Levin</surname><given-names>AM</given-names></name><name><surname>Thomas</surname><given-names>C</given-names></name><name><surname>Garcia</surname><given-names>KC</given-names></name></person-group><year>2012</year><article-title>Structural basis of Wnt recognition by Frizzled</article-title><source>Science</source><volume>337</volume><fpage>59</fpage><lpage>64</lpage><pub-id pub-id-type="doi">10.1126/science.1222879</pub-id></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Johnson</surname><given-names>JS</given-names></name><name><surname>Meliton</surname><given-names>V</given-names></name><name><surname>Kim</surname><given-names>WK</given-names></name><name><surname>Lee</surname><given-names>KB</given-names></name><name><surname>Wang</surname><given-names>JC</given-names></name><name><surname>Nguyen</surname><given-names>K</given-names></name><etal/></person-group><year>2011</year><article-title>Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo</article-title><source>J Cell Biochem</source><volume>112</volume><fpage>1673</fpage><lpage>84</lpage><pub-id pub-id-type="doi">10.1002/jcb.23082</pub-id></element-citation></ref><ref id="bib33"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kabsch</surname><given-names>W</given-names></name></person-group><year>2010</year><article-title>XDS</article-title><source>Acta Crystallogr D Biol Crystallogr</source><volume>66</volume><fpage>125</fpage><lpage>32</lpage><pub-id pub-id-type="doi">10.1107/S0907444909047337</pub-id></element-citation></ref><ref id="bib34"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kha</surname><given-names>HT</given-names></name><name><surname>Basseri</surname><given-names>B</given-names></name><name><surname>Shouhed</surname><given-names>D</given-names></name><name><surname>Richardson</surname><given-names>J</given-names></name><name><surname>Tetradis</surname><given-names>S</given-names></name><name><surname>Hahn</surname><given-names>TJ</given-names></name><etal/></person-group><year>2004</year><article-title>Oxysterols regulate differentiation of mesenchymal stem cells: pro-bone and anti-fat</article-title><source>J Bone Miner Res</source><volume>19</volume><fpage>830</fpage><lpage>40</lpage><pub-id pub-id-type="doi">10.1359/jbmr.040115</pub-id></element-citation></ref><ref id="bib35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kim</surname><given-names>J</given-names></name><name><surname>Tang</surname><given-names>JY</given-names></name><name><surname>Gong</surname><given-names>R</given-names></name><name><surname>Kim</surname><given-names>J</given-names></name><name><surname>Lee</surname><given-names>JJ</given-names></name><name><surname>Clemons</surname><given-names>KV</given-names></name><etal/></person-group><year>2010</year><article-title>Itraconazole, a commonly used antifungal that inhibits Hedgehog pathway activity and cancer growth</article-title><source>Cancer Cell</source><volume>17</volume><fpage>388</fpage><lpage>99</lpage><pub-id pub-id-type="doi">10.1016/j.ccr.2010.02.027</pub-id></element-citation></ref><ref id="bib36"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kim</surname><given-names>MS</given-names></name><name><surname>Saunders</surname><given-names>AM</given-names></name><name><surname>Hamaoka</surname><given-names>BY</given-names></name><name><surname>Beachy</surname><given-names>PA</given-names></name><name><surname>Leahy</surname><given-names>DJ</given-names></name></person-group><year>2011</year><article-title>Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling</article-title><source>Proc Natl Acad Sci USA</source><volume>108</volume><fpage>13112</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1073/pnas.1109877108</pub-id></element-citation></ref><ref id="bib37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kim</surname><given-names>WK</given-names></name><name><surname>Meliton</surname><given-names>V</given-names></name><name><surname>Amantea</surname><given-names>CM</given-names></name><name><surname>Hahn</surname><given-names>TJ</given-names></name><name><surname>Parhami</surname><given-names>F</given-names></name></person-group><year>2007</year><article-title>20(S)-hydroxycholesterol inhibits PPARgamma expression and adipogenic differentiaion of bone marrow stromal cells through a hedgehog-dependent mechanism</article-title><source>J Bone Miner Res</source><volume>22</volume><fpage>1711</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1359/jbmr.070710</pub-id></element-citation></ref><ref id="bib38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kwon</surname><given-names>HJ</given-names></name><name><surname>Abi-Mosleh</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>ML</given-names></name><name><surname>Deisenhofer</surname><given-names>J</given-names></name><name><surname>Goldstein</surname><given-names>JL</given-names></name><name><surname>Brown</surname><given-names>MS</given-names></name><etal/></person-group><year>2009</year><article-title>Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol</article-title><source>Cell</source><volume>137</volume><fpage>1213</fpage><lpage>24</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2009.03.049</pub-id></element-citation></ref><ref id="bib39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Larkin</surname><given-names>MA</given-names></name><name><surname>Blackshields</surname><given-names>G</given-names></name><name><surname>Brown</surname><given-names>NP</given-names></name><name><surname>Chenna</surname><given-names>R</given-names></name><name><surname>McGettigan</surname><given-names>PA</given-names></name><name><surname>McWilliam</surname><given-names>H</given-names></name><etal/></person-group><year>2007</year><article-title>Clustal W and Clustal X version 2.0</article-title><source>Bioinformatics</source><volume>23</volume><fpage>2947</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1093/bioinformatics/btm404</pub-id></element-citation></ref><ref id="bib40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lovell</surname><given-names>SC</given-names></name><name><surname>Word</surname><given-names>JM</given-names></name><name><surname>Richardson</surname><given-names>JS</given-names></name><name><surname>Richardson</surname><given-names>DC</given-names></name></person-group><year>2000</year><article-title>The penultimate rotamer library</article-title><source>Proteins</source><volume>40</volume><fpage>389</fpage><lpage>408</lpage><pub-id pub-id-type="doi">10.1002/1097-0134(20000815)40:3<389::AID-PROT50>3.0.CO;2-2</pub-id></element-citation></ref><ref id="bib41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Machold</surname><given-names>R</given-names></name><name><surname>Hayashi</surname><given-names>S</given-names></name><name><surname>Rutlin</surname><given-names>M</given-names></name><name><surname>Muzumdar</surname><given-names>MD</given-names></name><name><surname>Nery</surname><given-names>S</given-names></name><name><surname>Corbin</surname><given-names>JG</given-names></name><etal/></person-group><year>2003</year><article-title>Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches</article-title><source>Neuron</source><volume>39</volume><fpage>937</fpage><lpage>50</lpage><pub-id pub-id-type="doi">10.1016/S0896-6273(03)00561-0</pub-id></element-citation></ref><ref id="bib42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Marigo</surname><given-names>V</given-names></name><name><surname>Davey</surname><given-names>RA</given-names></name><name><surname>Zuo</surname><given-names>Y</given-names></name><name><surname>Cunningham</surname><given-names>JM</given-names></name><name><surname>Tabin</surname><given-names>CJ</given-names></name></person-group><year>1996</year><article-title>Biochemical evidence that patched is the Hedgehog receptor</article-title><source>Nature</source><volume>384</volume><fpage>176</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1038/384176a0</pub-id></element-citation></ref><ref id="bib43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Maurya</surname><given-names>AK</given-names></name><name><surname>Tan</surname><given-names>H</given-names></name><name><surname>Souren</surname><given-names>M</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name><name><surname>Wittbrodt</surname><given-names>J</given-names></name><name><surname>Ingham</surname><given-names>PW</given-names></name></person-group><year>2011</year><article-title>Integration of Hedgehog and BMP signalling by the engrailed2a gene in the zebrafish myotome</article-title><source>Development</source><volume>138</volume><fpage>755</fpage><lpage>65</lpage><pub-id pub-id-type="doi">10.1242/dev.062521</pub-id></element-citation></ref><ref id="bib44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mayo</surname><given-names>CJ</given-names></name><name><surname>Diprose</surname><given-names>JM</given-names></name><name><surname>Walter</surname><given-names>TS</given-names></name><name><surname>Berry</surname><given-names>IM</given-names></name><name><surname>Wilson</surname><given-names>J</given-names></name><name><surname>Owens</surname><given-names>RJ</given-names></name><etal/></person-group><year>2005</year><article-title>Benefits of automated crystallization plate tracking, imaging, and analysis</article-title><source>Structure</source><volume>13</volume><fpage>175</fpage><lpage>82</lpage><pub-id pub-id-type="doi">10.1016/j.str.2004.12.010</pub-id></element-citation></ref><ref id="bib45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McCoy</surname><given-names>AJ</given-names></name><name><surname>Grosse-Kunstleve</surname><given-names>RW</given-names></name><name><surname>Adams</surname><given-names>PD</given-names></name><name><surname>Winn</surname><given-names>MD</given-names></name><name><surname>Storoni</surname><given-names>LC</given-names></name><name><surname>Read</surname><given-names>RJ</given-names></name></person-group><year>2007</year><article-title>Phaser crystallographic software</article-title><source>J Appl Crystallogr</source><volume>40</volume><fpage>658</fpage><lpage>74</lpage><pub-id pub-id-type="doi">10.1107/S0021889807021206</pub-id></element-citation></ref><ref id="bib46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McMahon</surname><given-names>AP</given-names></name><name><surname>Ingham</surname><given-names>PW</given-names></name><name><surname>Tabin</surname><given-names>CJ</given-names></name></person-group><year>2003</year><article-title>Developmental roles and clinical significance of hedgehog signaling</article-title><source>Curr Top Dev Biol</source><volume>53</volume><fpage>1</fpage><lpage>114</lpage><pub-id pub-id-type="doi">10.1016/S0070-2153(03)53002-2</pub-id></element-citation></ref><ref id="bib47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mijares</surname><given-names>A</given-names></name><name><surname>Cargill</surname><given-names>DI</given-names></name><name><surname>Glasel</surname><given-names>JA</given-names></name><name><surname>Lieberman</surname><given-names>S</given-names></name></person-group><year>1967</year><article-title>Studies on C-20 epimers of 20-Hydroxycholesterol</article-title><source>J Org Chem</source><volume>32</volume><fpage>810</fpage><lpage>2</lpage><pub-id pub-id-type="doi">10.1021/jo01278a066</pub-id></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Molday</surname><given-names>RS</given-names></name><name><surname>MacKenzie</surname><given-names>D</given-names></name></person-group><year>1983</year><article-title>Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes</article-title><source>Biochemistry</source><volume>22</volume><fpage>653</fpage><lpage>60</lpage><pub-id pub-id-type="doi">10.1021/bi00272a020</pub-id></element-citation></ref><ref id="bib49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Monaco</surname><given-names>HL</given-names></name></person-group><year>1997</year><article-title>Crystal Structure of the chicken riboflavin-binding protein</article-title><source>EMBO J</source><volume>16</volume><fpage>1475</fpage><lpage>83</lpage><pub-id pub-id-type="doi">10.1093/emboj/16.7.1475</pub-id></element-citation></ref><ref id="bib50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Morris</surname><given-names>GM</given-names></name><name><surname>Huey</surname><given-names>R</given-names></name><name><surname>Lindstrom</surname><given-names>W</given-names></name><name><surname>Sanner</surname><given-names>MF</given-names></name><name><surname>Belew</surname><given-names>RK</given-names></name><name><surname>Goodsell</surname><given-names>DS</given-names></name><etal/></person-group><year>2009</year><article-title>AutoDock4 and AutoDockTools4: automated docking with Selective receptor flexibility</article-title><source>J Comput Chem</source><volume>30</volume><fpage>2785</fpage><lpage>91</lpage><pub-id pub-id-type="doi">10.1002/jcc.21256</pub-id></element-citation></ref><ref id="bib51"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Murone</surname><given-names>M</given-names></name><name><surname>Rosenthal</surname><given-names>A</given-names></name><name><surname>de Sauvage</surname><given-names>FJ</given-names></name></person-group><year>1999</year><article-title>Sonic hedgehog signaling by the patched-smoothened receptor complex</article-title><source>Curr Biol</source><volume>9</volume><fpage>76</fpage><lpage>84</lpage><pub-id pub-id-type="doi">10.1016/S0960-9822(99)80018-9</pub-id></element-citation></ref><ref id="bib52"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Murshudov</surname><given-names>GN</given-names></name><name><surname>Vagin</surname><given-names>AA</given-names></name><name><surname>Dodson</surname><given-names>EJ</given-names></name></person-group><year>1997</year><article-title>Refinement of macromolecular structures by the maximum-likelihood method</article-title><source>Acta Crystallogr D Biol Crystallogr</source><volume>53</volume><fpage>240</fpage><lpage>55</lpage><pub-id pub-id-type="doi">10.1107/S0907444996012255</pub-id></element-citation></ref><ref id="bib53"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nachtergaele</surname><given-names>S</given-names></name><name><surname>Mydock</surname><given-names>LK</given-names></name><name><surname>Krishnan</surname><given-names>K</given-names></name><name><surname>Rammohan</surname><given-names>J</given-names></name><name><surname>Schlesinger</surname><given-names>PH</given-names></name><name><surname>Covey</surname><given-names>DF</given-names></name><etal/></person-group><year>2012</year><article-title>Oxysterols are allosteric activators of the oncoprotein Smoothened</article-title><source>Nat Chem Biol</source><volume>8</volume><fpage>211</fpage><lpage>20</lpage><pub-id pub-id-type="doi">10.1038/nchembio.765</pub-id></element-citation></ref><ref id="bib54"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nakano</surname><given-names>Y</given-names></name><name><surname>Nystedt</surname><given-names>S</given-names></name><name><surname>Shivdasani</surname><given-names>AA</given-names></name><name><surname>Strutt</surname><given-names>H</given-names></name><name><surname>Thomas</surname><given-names>C</given-names></name><name><surname>Ingham</surname><given-names>PW</given-names></name></person-group><year>2004</year><article-title>Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila</article-title><source>Mech of Dev</source><volume>121</volume><fpage>507</fpage><lpage>18</lpage><pub-id pub-id-type="doi">10.1016/j.mod.2004.04.015</pub-id></element-citation></ref><ref id="bib55"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nedelcu</surname><given-names>D</given-names></name><name><surname>Liu</surname><given-names>J</given-names></name><name><surname>Xu</surname><given-names>Y</given-names></name><name><surname>Jao</surname><given-names>C</given-names></name><name><surname>Salic</surname><given-names>A</given-names></name></person-group><year>2013</year><article-title>Oxysterol binding to the extracellular domain of Smoothened in Hedgehog signaling</article-title><source>Nat Chem Biol</source><volume>9</volume><fpage>557</fpage><lpage>64</lpage><pub-id pub-id-type="doi">10.1038/nchembio.1290</pub-id></element-citation></ref><ref id="bib56"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Otwinowski</surname><given-names>Z</given-names></name><name><surname>Minor</surname><given-names>W</given-names></name></person-group><year>1997</year><article-title>Processing of x-ray diffraction data collected in Oscillation Mode</article-title><source>Methods Enzymol</source><volume>276</volume><fpage>307</fpage><lpage>26</lpage><pub-id pub-id-type="doi">10.1016/s0076-6879(97)76066-x</pub-id></element-citation></ref><ref id="bib57"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Oxtoby</surname><given-names>E</given-names></name><name><surname>Jowett</surname><given-names>T</given-names></name></person-group><year>1993</year><article-title>Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development</article-title><source>Nucleic Acids Res</source><volume>21</volume><fpage>1087</fpage><lpage>95</lpage><pub-id pub-id-type="doi">10.1093/nar/21.5.1087</pub-id></element-citation></ref><ref id="bib58"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pei</surname><given-names>J</given-names></name><name><surname>Grishin</surname><given-names>NV</given-names></name></person-group><year>2012</year><article-title>Cysteine-rich domains related to Frizzled receptors and Hedgehog-interacting proteins</article-title><source>Protein Sci</source><volume>21</volume><fpage>1172</fpage><lpage>84</lpage><pub-id pub-id-type="doi">10.1002/pro.2105</pub-id></element-citation></ref><ref id="bib59"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Phizicky</surname><given-names>EM</given-names></name><name><surname>Fields</surname><given-names>S</given-names></name></person-group><year>1995</year><article-title>Protein-protein interactions: methods for detection and analysis</article-title><source>Microbiol Rev</source><volume>59</volume><fpage>94</fpage><lpage>123</lpage></element-citation></ref><ref id="bib60"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pore</surname><given-names>VS</given-names></name><name><surname>Aher</surname><given-names>NG</given-names></name><name><surname>Kumar</surname><given-names>M</given-names></name><name><surname>Shukla</surname><given-names>PK</given-names></name></person-group><year>2006</year><article-title>Design and synthesis of fluconazole/bile acid conjugate using click reaction</article-title><source>Tetrahedron</source><volume>62</volume><fpage>11178</fpage><lpage>86</lpage><pub-id pub-id-type="doi">10.1016/j.tet.2006.09.021</pub-id></element-citation></ref><ref id="bib61"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Riese</surname><given-names>DJ</given-names><suffix>II</suffix></name></person-group><year>2011</year><article-title>Ligand-based receptor tyrosine kinase partial agonists: new paradigm for cancer drug discovery?</article-title><source>Expert Opin Drug Discov</source><volume>6</volume><fpage>185</fpage><lpage>93</lpage><pub-id pub-id-type="doi">10.1517/17460441.2011.547468</pub-id></element-citation></ref><ref id="bib62"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Riffel</surname><given-names>N</given-names></name><name><surname>Harlos</surname><given-names>K</given-names></name><name><surname>Iourin</surname><given-names>O</given-names></name><name><surname>Rao</surname><given-names>ZH</given-names></name><name><surname>Kingsman</surname><given-names>A</given-names></name><name><surname>Stuart</surname><given-names>D</given-names></name><etal/></person-group><year>2002</year><article-title>Atomic resolution structure of Moloney murine leukemia virus matrix protein and its relationship to other retroviral matrix proteins</article-title><source>Structure</source><volume>10</volume><fpage>1627</fpage><lpage>36</lpage><pub-id pub-id-type="doi">10.1016/S0969-2126(02)00896-1</pub-id></element-citation></ref><ref id="bib63"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Robarge</surname><given-names>KD</given-names></name><name><surname>Brunton</surname><given-names>SA</given-names></name><name><surname>Castanedo</surname><given-names>GM</given-names></name><name><surname>Cui</surname><given-names>Y</given-names></name><name><surname>Dina</surname><given-names>MS</given-names></name><name><surname>Goldsmith</surname><given-names>R</given-names></name><etal/></person-group><year>2009</year><article-title>GDC-0449—a potent inhibitor of the hedgehog pathway</article-title><source>Bioorg Med Chem Lett</source><volume>19</volume><fpage>5576</fpage><lpage>81</lpage><pub-id pub-id-type="doi">10.1016/j.bmcl.2009.08.049</pub-id></element-citation></ref><ref id="bib64"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rohatgi</surname><given-names>R</given-names></name><name><surname>Milenkovic</surname><given-names>L</given-names></name><name><surname>Corcoran</surname><given-names>RB</given-names></name><name><surname>Scott</surname><given-names>MP</given-names></name></person-group><year>2009</year><article-title>Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process</article-title><source>Proc Natl Acad Sci USA</source><volume>106</volume><fpage>3196</fpage><lpage>201</lpage><pub-id pub-id-type="doi">10.1073/pnas.0813373106</pub-id></element-citation></ref><ref id="bib65"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rohatgi</surname><given-names>R</given-names></name><name><surname>Milenkovic</surname><given-names>L</given-names></name><name><surname>Scott</surname><given-names>MP</given-names></name></person-group><year>2007</year><article-title>Patched1 regulates hedgehog signaling at the primary cilium</article-title><source>Science</source><volume>317</volume><fpage>372</fpage><lpage>6</lpage><pub-id pub-id-type="doi">10.1126/science.1139740</pub-id></element-citation></ref><ref id="bib66"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rominger</surname><given-names>CM</given-names></name><name><surname>Bee</surname><given-names>WL</given-names></name><name><surname>Copeland</surname><given-names>RA</given-names></name><name><surname>Davenport</surname><given-names>EA</given-names></name><name><surname>Gilmartin</surname><given-names>A</given-names></name><name><surname>Gontarek</surname><given-names>R</given-names></name><etal/></person-group><year>2009</year><article-title>Evidence for allosteric interactions of antagonist binding to the smoothened receptor</article-title><source>J Pharmacol Exp Ther</source><volume>329</volume><fpage>995</fpage><lpage>1005</lpage><pub-id pub-id-type="doi">10.1124/jpet.109.152090</pub-id></element-citation></ref><ref id="bib67"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ryu</surname><given-names>EH</given-names></name><name><surname>Yan</surname><given-names>J</given-names></name><name><surname>Zhong</surname><given-names>Z</given-names></name><name><surname>Zhao</surname><given-names>Y</given-names></name></person-group><year>2006</year><article-title>Solvent-induced amphiphilic molecular baskets: Unimolecular reversed micelles with different size, shape, and flexibility</article-title><source>J Org Chem</source><volume>71</volume><fpage>7205</fpage><lpage>13</lpage><pub-id pub-id-type="doi">10.1021/jo0607663</pub-id></element-citation></ref><ref id="bib68"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sasaki</surname><given-names>H</given-names></name><name><surname>Hui</surname><given-names>C</given-names></name><name><surname>Nakafuku</surname><given-names>M</given-names></name><name><surname>Kondoh</surname><given-names>H</given-names></name></person-group><year>1997</year><article-title>A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro</article-title><source>Development</source><volume>124</volume><fpage>1313</fpage><lpage>22</lpage></element-citation></ref><ref id="bib69"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Scales</surname><given-names>SJ</given-names></name><name><surname>de Sauvage</surname><given-names>FJ</given-names></name></person-group><year>2009</year><article-title>Mechanisms of Hedgehog pathway activation in cancer and implications for therapy</article-title><source>Trends Pharmacol Sci</source><volume>30</volume><fpage>303</fpage><lpage>12</lpage><pub-id pub-id-type="doi">10.1016/j.tips.2009.03.007</pub-id></element-citation></ref><ref id="bib70"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schneider</surname><given-names>TR</given-names></name><name><surname>Sheldrick</surname><given-names>GM</given-names></name></person-group><year>2002</year><article-title>Substructure solution with SHELXD</article-title><source>Acta Crystallogr D Biol Crystallogr</source><volume>58</volume><fpage>1772</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1107/S0907444902011678</pub-id></element-citation></ref><ref id="bib71"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sharpe</surname><given-names>HJ</given-names></name><name><surname>de Sauvage</surname><given-names>FJ</given-names></name></person-group><year>2012</year><article-title>Signaling: an oxysterol ligand for Smoothened</article-title><source>Nat Chem Biol</source><volume>8</volume><fpage>139</fpage><lpage>40</lpage><pub-id pub-id-type="doi">10.1038/nchembio.774</pub-id></element-citation></ref><ref id="bib72"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shaw</surname><given-names>PE</given-names></name></person-group><year>1966</year><article-title>17beta Alkylation of a 17-Keto steroid by Alkylmagnesium Halides</article-title><source>J Org Chem</source><volume>31</volume><fpage>2119</fpage><lpage>24</lpage><pub-id pub-id-type="doi">10.1021/jo01345a016</pub-id></element-citation></ref><ref id="bib73"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shin</surname><given-names>K</given-names></name><name><surname>Lee</surname><given-names>J</given-names></name><name><surname>Guo</surname><given-names>N</given-names></name><name><surname>Kim</surname><given-names>J</given-names></name><name><surname>Lim</surname><given-names>A</given-names></name><name><surname>Qu</surname><given-names>L</given-names></name><etal/></person-group><year>2011</year><article-title>Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder</article-title><source>Nature</source><volume>472</volume><fpage>110</fpage><lpage>4</lpage><pub-id pub-id-type="doi">10.1038/nature09851</pub-id></element-citation></ref><ref id="bib74"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stiegler</surname><given-names>AL</given-names></name><name><surname>Burden</surname><given-names>SJ</given-names></name><name><surname>Hubbard</surname><given-names>SR</given-names></name></person-group><year>2009</year><article-title>Crystal structure of the frizzled-like cysteine-rich domain of the receptor tyrosine kinase MuSK</article-title><source>J Mol Biol</source><volume>393</volume><fpage>1</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1016/j.jmb.2009.07.091</pub-id></element-citation></ref><ref id="bib75"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stone</surname><given-names>DM</given-names></name><name><surname>Hynes</surname><given-names>M</given-names></name><name><surname>Armanini</surname><given-names>M</given-names></name><name><surname>Swanson</surname><given-names>TA</given-names></name><name><surname>Gu</surname><given-names>Q</given-names></name><name><surname>Johnson</surname><given-names>RL</given-names></name><etal/></person-group><year>1996</year><article-title>The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog</article-title><source>Nature</source><volume>384</volume><fpage>129</fpage><lpage>34</lpage><pub-id pub-id-type="doi">10.1038/384129a0</pub-id></element-citation></ref><ref id="bib76"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stuart</surname><given-names>DI</given-names></name><name><surname>Levine</surname><given-names>M</given-names></name><name><surname>Muirhead</surname><given-names>H</given-names></name><name><surname>Stammers</surname><given-names>DK</given-names></name></person-group><year>1979</year><article-title>Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 Å</article-title><source>J Mol Biol</source><volume>134</volume><fpage>109</fpage><lpage>42</lpage><pub-id pub-id-type="doi">10.1016/0022-2836(79)90416-9</pub-id></element-citation></ref><ref id="bib77"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Svensson</surname><given-names>G</given-names></name><name><surname>Awad</surname><given-names>W</given-names></name><name><surname>Håkansson</surname><given-names>M</given-names></name><name><surname>Mani</surname><given-names>K</given-names></name><name><surname>Logan</surname><given-names>DT</given-names></name></person-group><year>2012</year><article-title>Crystal structure of N-glycosylated human glypican-1 core protein: structure of two loops evolutionarily conserved in vertebrate glypican-1</article-title><source>J Biol Chem</source><volume>287</volume><fpage>14040</fpage><lpage>51</lpage><pub-id pub-id-type="doi">10.1074/jbc.M111.322487</pub-id></element-citation></ref><ref id="bib78"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Taipale</surname><given-names>J</given-names></name><name><surname>Chen</surname><given-names>JK</given-names></name><name><surname>Cooper</surname><given-names>MK</given-names></name><name><surname>Wang</surname><given-names>B</given-names></name><name><surname>Mann</surname><given-names>RK</given-names></name><name><surname>Milenkovic</surname><given-names>L</given-names></name><etal/></person-group><year>2000</year><article-title>Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine</article-title><source>Nature</source><volume>406</volume><fpage>1005</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1038/35023008</pub-id></element-citation></ref><ref id="bib79"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Taipale</surname><given-names>J</given-names></name><name><surname>Cooper</surname><given-names>MK</given-names></name><name><surname>Maiti</surname><given-names>T</given-names></name><name><surname>Beachy</surname><given-names>PA</given-names></name></person-group><year>2002</year><article-title>Patched acts catalytically to suppress the activity of Smoothened</article-title><source>Nature</source><volume>418</volume><fpage>892</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1038/nature00989</pub-id></element-citation></ref><ref id="bib80"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tang</surname><given-names>JY</given-names></name><name><surname>Mackay-Wiggan</surname><given-names>JM</given-names></name><name><surname>Aszterbaum</surname><given-names>M</given-names></name><name><surname>Yauch</surname><given-names>RL</given-names></name><name><surname>Lindgren</surname><given-names>J</given-names></name><name><surname>Chang</surname><given-names>K</given-names></name><etal/></person-group><year>2012</year><article-title>Inhibiting the hedgehog pathway in patients with the basal-cell nevus syndrome</article-title><source>N Engl J Med</source><volume>366</volume><fpage>2180</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1056/NEJMoa1113538</pub-id></element-citation></ref><ref id="bib81"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Terwilliger</surname><given-names>TC</given-names></name></person-group><year>2003</year><article-title>SOLVE and RESOLVE: automated structure solution and density modification</article-title><source>Methods Enzymol</source><volume>374</volume><fpage>22</fpage><lpage>37</lpage><pub-id pub-id-type="doi">10.1016/S0076-6879(03)74002-6</pub-id></element-citation></ref><ref id="bib82"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Varjosalo</surname><given-names>M</given-names></name><name><surname>Li</surname><given-names>SP</given-names></name><name><surname>Taipale</surname><given-names>J</given-names></name></person-group><year>2006</year><article-title>Divergence of hedgehog signal transduction mechanism between Drosophila and mammals</article-title><source>Dev Cell</source><volume>10</volume><fpage>177</fpage><lpage>86</lpage><pub-id pub-id-type="doi">10.1016/j.devcel.2005.12.014</pub-id></element-citation></ref><ref id="bib83"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Von Hoff</surname><given-names>DD</given-names></name><name><surname>LoRusso</surname><given-names>PM</given-names></name><name><surname>Rudin</surname><given-names>CM</given-names></name><name><surname>Reddy</surname><given-names>JC</given-names></name><name><surname>Yauch</surname><given-names>RL</given-names></name><name><surname>Tibes</surname><given-names>R</given-names></name><etal/></person-group><year>2009</year><article-title>Inhibition of the hedgehog pathway in advanced basal-cell carcinoma</article-title><source>N Engl J Med</source><volume>361</volume><fpage>1164</fpage><lpage>72</lpage><pub-id pub-id-type="doi">10.1056/NEJMoa0905360</pub-id></element-citation></ref><ref id="bib84"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Walter</surname><given-names>TS</given-names></name><name><surname>Diprose</surname><given-names>JM</given-names></name><name><surname>Mayo</surname><given-names>CJ</given-names></name><name><surname>Siebold</surname><given-names>C</given-names></name><name><surname>Pickford</surname><given-names>MG</given-names></name><name><surname>Carter</surname><given-names>L</given-names></name><etal/></person-group><year>2005</year><article-title>A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization</article-title><source>Acta Crystallogr D Biol Crystallogr</source><volume>61</volume><fpage>651</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1107/S0907444905007808</pub-id></element-citation></ref><ref id="bib85"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>C</given-names></name><name><surname>Wu</surname><given-names>H</given-names></name><name><surname>Katritch</surname><given-names>V</given-names></name><name><surname>Han</surname><given-names>GW</given-names></name><name><surname>Huang</surname><given-names>XP</given-names></name><name><surname>Liu</surname><given-names>W</given-names></name><etal/></person-group><year>2013</year><article-title>Structure of the human smoothened receptor bound to an antitumour agent</article-title><source>Nature</source><volume>497</volume><fpage>338</fpage><lpage>43</lpage><pub-id pub-id-type="doi">10.1038/nature12167</pub-id></element-citation></ref><ref id="bib86"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>ML</given-names></name><name><surname>Motamed</surname><given-names>M</given-names></name><name><surname>Infante</surname><given-names>RE</given-names></name><name><surname>Abi-Mosleh</surname><given-names>L</given-names></name><name><surname>Kwon</surname><given-names>HJ</given-names></name><name><surname>Brown</surname><given-names>MS</given-names></name><etal/></person-group><year>2010</year><article-title>Identification of surface residues on Niemann-Pick C2 essential for hydrophobic handoff of cholesterol to NPC1 in lysosomes</article-title><source>Cell Metab</source><volume>12</volume><fpage>166</fpage><lpage>73</lpage><pub-id pub-id-type="doi">10.1016/j.cmet.2010.05.016</pub-id></element-citation></ref><ref id="bib87"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Davidow</surname><given-names>L</given-names></name><name><surname>Arvanites</surname><given-names>AC</given-names></name><name><surname>Blanchard</surname><given-names>J</given-names></name><name><surname>Lam</surname><given-names>K</given-names></name><name><surname>Xu</surname><given-names>K</given-names></name><etal/></person-group><year>2012</year><article-title>Glucocorticoid compounds modify smoothened localization and hedgehog pathway activity</article-title><source>Chem Biol</source><volume>9</volume><fpage>972</fpage><lpage>82</lpage><pub-id pub-id-type="doi">10.1016/j.chembiol.2012.06.012</pub-id></element-citation></ref><ref id="bib88"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>YL</given-names></name><name><surname>Xiao</surname><given-names>JW</given-names></name><name><surname>Suzek</surname><given-names>TO</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>JY</given-names></name><name><surname>Bryant</surname><given-names>SH</given-names></name></person-group><year>2009</year><article-title>PubChem: a public information system for analyzing bioactivities of small molecules</article-title><source>Nucleic Acids Res</source><volume>37</volume><fpage>W623</fpage><lpage>33</lpage><pub-id pub-id-type="doi">10.1093/nar/gkp456</pub-id></element-citation></ref><ref id="bib89"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Winter</surname><given-names>G</given-names></name></person-group><year>2010</year><article-title>XIA2: an expert system for macromolecular crystallography data reduction</article-title><source>J Appl Crystallogr</source><volume>43</volume><fpage>186</fpage><lpage>90</lpage><pub-id pub-id-type="doi">10.1107/S0021889809045701</pub-id></element-citation></ref><ref id="bib90"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wolff</surname><given-names>C</given-names></name><name><surname>Roy</surname><given-names>S</given-names></name><name><surname>Ingham</surname><given-names>PW</given-names></name></person-group><year>2003</year><article-title>Multiple muscle cell identities induced by distinct levels and timing of Hedgehog activity in the zebrafish embryo</article-title><source>Curr Biol</source><volume>13</volume><fpage>1169</fpage><lpage>81</lpage><pub-id pub-id-type="doi">10.1016/S0960-9822(03)00461-5</pub-id></element-citation></ref><ref id="bib91"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yauch</surname><given-names>RL</given-names></name><name><surname>Dijkgraaf</surname><given-names>GJ</given-names></name><name><surname>Alicke</surname><given-names>B</given-names></name><name><surname>Januario</surname><given-names>T</given-names></name><name><surname>Ahn</surname><given-names>CP</given-names></name><name><surname>Holcomb</surname><given-names>T</given-names></name><etal/></person-group><year>2009</year><article-title>Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma</article-title><source>Science</source><volume>326</volume><fpage>572</fpage><lpage>4</lpage><pub-id pub-id-type="doi">10.1126/science.1179386</pub-id></element-citation></ref><ref id="bib92"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhao</surname><given-names>Y</given-names></name><name><surname>Bishop</surname><given-names>B</given-names></name><name><surname>Clay</surname><given-names>JE</given-names></name><name><surname>Lu</surname><given-names>W</given-names></name><name><surname>Jones</surname><given-names>M</given-names></name><name><surname>Daenke</surname><given-names>S</given-names></name><etal/></person-group><year>2011</year><article-title>Automation of large scale transient protein expression in mammalian cells</article-title><source>J Struct Biol</source><volume>175</volume><fpage>209</fpage><lpage>15</lpage><pub-id pub-id-type="doi">10.1016/j.jsb.2011.04.017</pub-id></element-citation></ref><ref id="bib93"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhao</surname><given-names>Y</given-names></name><name><surname>Zhong</surname><given-names>ZQ</given-names></name></person-group><year>2005</year><article-title>Oligomeric cholates: amphiphilic foldamers with nanometer-sized hydrophilic cavities</article-title><source>J Am Chem Soc</source><volume>127</volume><fpage>17894</fpage><lpage>901</lpage><pub-id pub-id-type="doi">10.1021/ja056151p</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01340.019</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Kuriyan</surname><given-names>John</given-names></name><role>Reviewing editor</role><aff><institution>Howard Hughes Medical Institute, University of California, Berkeley</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elife.elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Reviewing editor and 2 reviewers.</p><p>The Reviewing editor and the two reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission. We anticipate that it will be straightforward to respond to these comments in a revised manuscript and, assuming that you are able to do so, you will receive a quick final decision from the editor without involving the reviewers any further.</p><p>The Hedgehog signaling pathway is important for tissue development and injury responses, and misregulation of this pathway can lead to cancer. Nachtergaele et al. build on previous work from their and other laboratories showing the ability of specific hydroxysterols to activate the Hedgehog signaling pathway through interactions with the 7TM protein Smoothened (Smo). In this new work, Nachtergaele and colleagues show that (i) hydroxysterols interact specifically with the N-terminal, extracellular cysteine-rich domain (CRD) of Smo (and their binding site is thus distinct from the well-characterized antagonist/agonist binding site in the 7 TM region), (ii) hydroxysterols interact with the Smo CRD at a site that homologous to the site on Frizzled CRDs that interacts with the palmitoleyl group attached to Wnt, (iii) hydroxysterol analogs can be created that show partial inhibition of Hedgehog signaling and hold promise as Hedgehog pathway-targeting therapeutics, (iv) interactions between Smo CRDs and hydroxysterols appear to be specific to vertebrate Smos, (v) hydroxysterols can activate Hedgehog signaling in fish embryos, and (vi) determine the 2.3 Å crystal structure of the Smo CRD from zebrafish.</p><p>These results are persuasive, well described, and represent significant advances in our understanding of how small molecules regulate Smo behavior. The Salic group recently published similar results in Nature Chemical Biology and we are aware of a paper in press at Developmental Cell also reporting similar results. There is overlap in many of the experiments and conclusions mapping the oxysterol binding domains of vertebrate Smoothened, the relationship between oxysterol engagement here and the action of inhibitors and activators within the 7 TM domain, and the reasons why <italic>Drosophila</italic> Smoothened works differently. Actual structural data on the CRD for Smoothened rather than inferred structural information in the Nedelcu based on lipid interactions with related CRDs for Frizzled family members distinguishes the paper under review. However, an actual structure with the CRD bound to oxysterol is not presented. Nevertheless, that all of these authors come to similar conclusions strengthens the impact of their work, and given the importance and interest in this molecule and the pathway it regulates, having more than one excellent report on this topic is no bad thing. Further, there are interesting differences in the behavior of “intelligent” drugs developed from an oxysterol base that adds an intriguing element to the current work.</p><p>Please address the following issues in a revised manuscript:</p><p>1) The authors discuss the possibility that cyclopamine may engage both the CRD and 7 TM domain, explaining properties such as cyclopamine's ability to induce cilial trafficking while non-sterol pathway inhibitors thought to act at the 7 TM region do not have this effect. However, cyclopamine does not appear to compete with oxysterol binding to the CRD region in the authors' biochemical studies—a result at odds with this conclusion (<xref ref-type="fig" rid="fig7">Figure 7C</xref>). These data create confusion regarding the interplay the 7 TM and CRD sites). There are also recent data suggesting glucocorticoid interactions from Wang et al. that the authors might bring into the discussion. The authors are encouraged to open up some more in their discussion, making it very clear where their data agrees or disagrees with conclusions in Nedelcu et al., how they interpret disagreement and how they perceive the role of oxysterols in normal HH signaling.</p><p>2) There is an odd discrepancy in the figures that the authors should address. Firstly, in <xref ref-type="fig" rid="fig1">Figure 1C</xref> the Δ-CRD form of mSmo migrates higher on the gel than Wild-type mSmo when deletion of a domain suggests it should run lower (as it does in <xref ref-type="fig" rid="fig6">Figure 6E</xref>). Is there some mistake in the labeling or loading of the gel in <xref ref-type="fig" rid="fig1">Figure 1C</xref>? Also, in <xref ref-type="fig" rid="fig6">Figure 6E</xref> the blot is labeled anti-Fc, but these forms of mSmo do not appear to have been Fc-tagged (vs YFP tagged).</p><p>3) The description of the zebrafish embryo experiments (<xref ref-type="fig" rid="fig4">Figure 4B</xref>) is difficult to follow for a more general audience. Please provide a more complete explanation and motivation for this experiment. Because only one example of each condition is shown and there is no data quantitation, it is difficult to know if the control and oxysterol treated samples are significantly different.</p><p>4) It would be useful to describe the fitting procedure for the bead-binding/competition curves and include a table that outlines the relative affinities for the different constructs/compounds studied.</p><p>5) The MALS experiments were performed at a zSmo CRD concentration of 5 μM. This is a very low concentration, so it is difficult to definitively state that the protein is monomeric. MALS experiments at higher protein concentrations would be useful to support the authors’ conclusion, or the authors could just say that the protein does not dimerize at low concentration.</p><p>6) A description of the structural comparison of the zSmo CRD is lacking. It is unclear what methods the authors used to make these comparisons and generate a phylogenetic tree. Are there other uncharacterized receptors that belong to this family? What are the rms deviations for the core parts of the structure, and over how many residues?</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01340.020</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) The authors discuss the possibility that cyclopamine may engage both the CRD and 7 TM domain, explaining properties such as cyclopamine's ability to induce cilial trafficking while non-sterol pathway inhibitors thought to act atthe 7 TM region do not have this effect. However, cyclopamine does not appear to compete with oxysterol binding to the CRD region in the authors' biochemical studies—a result at odds with this conclusion (</italic><xref ref-type="fig" rid="fig7"><italic>Figure 7C</italic></xref><italic>). These data create confusion regarding the interplay the 7 TM and CRD sites</italic>.</p><p>We thank the reviewers for pointing out the potential contradiction between the results presented <xref ref-type="fig" rid="fig7 fig8">Figures 7C and 8G</xref>. <xref ref-type="fig" rid="fig8">Figure 8G</xref> is a ligand affinity chromatography assay demonstrating that cyclopamine can indeed inhibit binding of the isolated Smo CRD-Fc to 20(S)-OHC beads. This observation is the basis of our hypothesis that cyclopamine can engage both the CRD and the 7 TM domains. <xref ref-type="fig" rid="fig7">Figure 7C</xref> is an assay that measures binding of bodipy-conjugated cyclopamine to cells expressing full-length Smo. In this assay (as shown previously) 20(S)-OHC does not block binding of bodipy-cyclopamine to Smo-expressing cells. Thus, we detect competition between 20(S)-OHC and cyclopamine when using the isolated, purified Smo CRD but not when using cells expressing full-length Smo. We believe the difference is because the cell-binding assay is significantly less sensitive than the affinity chromatography assay and is largely reporting on the higher affinity interaction between cyclopamine and the 7 TM site (which cannot be competed with oxysterols). Alternatively, it is possible that cyclopamine binds the CRD more weakly when it is embedded in the context of the whole protein.</p><p>The distinction between binding to the isolated CRD protein (<xref ref-type="fig" rid="fig8">Figure 8G</xref>) and full-length Smo (<xref ref-type="fig" rid="fig7">Figure 7C</xref>) in cells is now explicitly described in the Discussion, along with the potential explanations mentioned above. In terms of the interplay between the two sites, we describe the CRD and 7 TM sites as being physically distinct but allosterically linked early in the discussion.</p><p><italic>There are also recent data suggesting glucocorticoid interactions from Wang et al that the authors might bring into the discussion</italic>.</p><p>We agree that the Wang et al. paper is indeed quite relevant as it shows that Hh-active glucocorticoids fall into two distinct classes—a set that competes with cyclopamine and a set that does not—supporting the view that there are two ligand binding sites on Smo. This paper is now discussed and referenced both in the Results (in the context of our discussion on the oxysterol-based inhibitors) and in the Discussion.</p><p><italic>The authors are encouraged to open up some more in their discussion, making it very clear where their data agrees or disagrees with conclusions in Nedelcu et al., how they interpret disagreement and how they perceive the role of oxysterols in normal HH signaling</italic>.</p><p>As described in the response to point 1, there are two distinct assays that have been used by us and by Nedelcu et al. to measure competition—binding of the isolated CRD to oxysterol beads and binding of bodipy cyclopamine to Smo expressing cells. Our data (<xref ref-type="fig" rid="fig7">Figure 7C</xref>) are in agreement with Nedelcu et al. (and previous studies described in <xref ref-type="bibr" rid="bib24">Dwyer et al., 2007</xref>) that oxysterols do not compete with cyclopamine for binding to Smo-expressing cells. Nedelcu et al. did not report on whether cyclopamine competes with 20(S)-OHC for binding to the isolated Smo CRD—the experiment we show in <xref ref-type="fig" rid="fig8">Figure 8G</xref>—and thus there is no disagreement on this point.</p><p>We also now explicitly discuss (both in the Results and the Discussion) the following points of comparison between our manuscript and that of Nedelcu et al.:</p><p>A) The different properties of our CRD-targeted partial agonists of Smo compared to the CRD-targeted inhibitor from Nedelcu et al.</p><p>B) A comparison of Shh-responsiveness of the CRD-deleted Smo proteins reported in both the studies.</p><p>C) As the reviewers recommend, we have expanded the discussion to clarify our views on whether endogenous oxysterols regulate Hh signaling, whether oxysterols mediate the interaction between Ptch1 and Smo, and what specific studies will be required in the future to conclusively answer this important question.</p><p><italic>2) There is an odd discrepancy in the figures that the authors should address. Firstly, in</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1C</italic></xref> <italic>the Δ-CRD form of mSmo migrates higher on the gel than Wild-type mSmo when deletion of a domain suggests it should run lower (as it does in</italic> <xref ref-type="fig" rid="fig6"><italic>Figure 6E</italic></xref><italic>). Is there some mistake in the labeling or loading of the gel in</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1C</italic></xref><italic>? Also, in</italic> <xref ref-type="fig" rid="fig6"><italic>Figure 6E</italic></xref> <italic>the blot is labeled anti-Fc, but these forms of mSmo do not appear to have been Fc-tagged (vs YFP tagged)</italic>.</p><p>We consistently find that YFP-Smo and ΔCRD-YFP-Smo migrate at roughly the same position when fractionated on 8% (37.5:1 Acrylamide:Bis) SDS-PAGE gels run using an alkaline (pH=8.8) Tris-Glycine buffer system. This is seen in <xref ref-type="fig" rid="fig1">Figure 1C</xref> and again in <xref ref-type="fig" rid="fig3">Figure 3A</xref>, which depicts lysates from stable cell lines expressing YFP-Smo and ΔCRD-YFP-Smo. However, ΔCRD-YFP-Smo runs lower than YFP-Smo (as expected) when fractionated on a 4–12% gradient gel run using a neutral (Bis-Tris, pH=7.0) buffer system, which is used in <xref ref-type="fig" rid="fig6">Figure 6E</xref>. Thus, the gel matrix composition and buffer system used had a large effect on the relative migration of YFP-Smo and ΔCRD-YFP-Smo. The different gel electrophoresis conditions are now noted in the respective figure legends.</p><p>Figure 6E is indeed an anti-YFP immunoblot (and not an anti-Fc immunoblot). We apologize for this error and have corrected it in the revised manuscript.</p><p><italic>3) The description of the zebrafish embryo experiments (</italic><xref ref-type="fig" rid="fig4"><italic>Figure 4B</italic></xref><italic>) is difficult to follow for a more general audience. Please provide a more complete explanation and motivation for this experiment. Because only one example of each condition is shown and there is no data quantitation, it is difficult to know if the control and oxysterol treated samples are significantly different</italic>.</p><p>We have now expanded the description of the experiment and its motivation in the Results. The main motivation for the experiment is to demonstrate that zebrafish Smo (whose CRD we have crystallized and characterized in biochemical assays) does in fact respond to oxysterols. Since full length zebrafish Smo is poorly expressed in mammalian cells, we treated embryos to determine if endogenous Smo can respond to oxysterols by activating a transgenic Hh pathway reporter used to study Hh signaling during zebrafish development. In addition, data quantitation and statistical analysis of significance for this experiment is now provided in a new figure (Figure 4–figure supplement 1).</p><p><italic>4) It would be useful to describe the fitting procedure for the bead-binding/competition curves and include a table that outlines the relative affinities for the different constructs/compounds studied</italic>.</p><p>The fitting procedures and equations for all curves, shown in both the binding and signaling assays, are now included in the Methods section. For the binding experiments, two proteins were used—the mouse Smo CRD-Fc and the zebrafish Smo ECD. We have included saturation curves for the binding of both proteins to 20(S)-OHC beads in <xref ref-type="fig" rid="fig2">Figure 2B</xref> (new in the revised manuscript) and 4D, respectively. Estimates of the dissociation constants for the binding of these two proteins to 20(S)-OHC beads are noted in the respective figure legends. We have not used competition curves to derive dissociation constants because we measured the ability of free ligand to block binding of the Smo CRD to bead immobilized ligand—a condition in which the two ligands are clearly chemically different (hence violating a key assumption when using competition to derive dissociation constants). Finally, Figure 8–figure supplement 1 has been added to summarize the IC50 values for the two oxysterol-based inhibitors, 20(R)-yne and 20-keto-yne.</p><p><italic>5) The MALS experiments were performed at a zSmo CRD concentration of 5 μM. This is a very low concentration, so it is difficult to definitively state that the protein is monomeric. MALS experiments at higher protein concentrations would be useful to support the authors’ conclusion, or the authors could just say that the protein does not dimerize at low concentration</italic>.</p><p>We have clarified the text as suggested and changed “In agreement with this crystal packing analysis, purified zSmo ectodomain behaved as a monomer in solution at concentrations up to 5 μM when assessed using multi angle light scattering (Figure 5–figure supplement 1D)” to “In agreement with this crystal packing analysis, purified zSmo ectodomain behaved as a monomer in solution at low concentrations (5 μM) when assessed using multi angle light scattering (Figure 5–figure supplement 1D).”</p><p><italic>6) A description of the structural comparison of the zSmo CRD is lacking. It is unclear what methods the authors used to make these comparisons and generate a phylogenetic tree</italic>.</p><p>We apologize for not being clearer in our description of the structural phylogenetic tree analysis. We have now included a description in the Methods and replaced “Structure-based phylogenetic analysis of the CRD domains was performed using SHP (<xref ref-type="bibr" rid="bib62">Riffel et al., 2002</xref>)”with “Superposition of CRD structures and root mean square deviation (RMSD) values were calculated for equivalent Cα atoms using program SHP (<xref ref-type="bibr" rid="bib76">Stuart et al., 1979</xref>; <xref ref-type="bibr" rid="bib62">Riffel et al., 2002</xref>). The phylogenetic tree for CRDs (<xref ref-type="fig" rid="fig5">Figure 5B</xref>) was prepared with program PHYLIP (<xref ref-type="bibr" rid="bib28">Felsenstein, 1989</xref>) with the summed structural correlation data presented in Figure 5–figure supplement 3 to construct a distance matrix.”</p><p>In addition, we changed the title of the Methods section from “Structure determination and refinement of zSmo ectodomain” to “Structure determination, refinement and analyses of the zSmo ectodomain.”</p><p><italic>Are there other uncharacterized receptors that belong to this family</italic>?</p><p>We thank the reviewers for this comment. Indeed, CRDs are structurally related distantly to the N-lobe region of the glypican protein family (<xref ref-type="bibr" rid="bib58">Pei and Grishin, 2012</xref>; <xref ref-type="bibr" rid="bib36">Kim et al., 2011</xref>; <xref ref-type="bibr" rid="bib77">Svensson et al., 2012</xref>). However, the N-lobe does not contain a small molecule binding groove or pocket and many of the Fz/Smo CRD motifs are different (<xref ref-type="bibr" rid="bib58">Pei and Grishin, 2012</xref>); thus, we have not included glypicans in our phylogenetic analysis. However, we have now included a note about glypicans in the legend of <xref ref-type="fig" rid="fig5">Figure 5B</xref>.</p><p><italic>What are the rms deviations for the core parts of the structure, and over how many residues</italic>?</p><p>As suggested, we have now included RMSD values and number of equivalent Cα atoms for the structural comparison of the CRDs in the new Figure 5–figure supplement 3.</p></body></sub-article></article> |