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">01808</article-id><article-id pub-id-type="doi">10.7554/eLife.01808</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Biophysics and structural biology</subject></subj-group></article-categories><title-group><article-title>Phosphoinositide-mediated oligomerization of a defensin induces cell lysis</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-8899"><name><surname>Poon</surname><given-names>Ivan KH</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf4"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-8900"><name><surname>Baxter</surname><given-names>Amy A</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf4"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-8901"><name><surname>Lay</surname><given-names>Fung T</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf4"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-8902"><name><surname>Mills</surname><given-names>Grant D</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf4"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-8903"><name><surname>Adda</surname><given-names>Christopher G</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf4"/></contrib><contrib contrib-type="author" id="author-8904"><name><surname>Payne</surname><given-names>Jennifer AE</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf4"/></contrib><contrib contrib-type="author" id="author-8905"><name><surname>Phan</surname><given-names>Thanh Kha</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf4"/></contrib><contrib contrib-type="author" id="author-8906"><name><surname>Ryan</surname><given-names>Gemma F</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf4"/></contrib><contrib contrib-type="author" id="author-8907"><name><surname>White</surname><given-names>Julie A</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con11"/><xref ref-type="fn" rid="conf4"/></contrib><contrib contrib-type="author" id="author-8908"><name><surname>Veneer</surname><given-names>Prem K</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con12"/><xref ref-type="fn" rid="conf4"/></contrib><contrib contrib-type="author" id="author-8909"><name><surname>van der Weerden</surname><given-names>Nicole L</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con13"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-8910"><name><surname>Anderson</surname><given-names>Marilyn A</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con14"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" corresp="yes" id="author-8912"><name><surname>Kvansakul</surname><given-names>Marc</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf4"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-4886"><name><surname>Hulett</surname><given-names>Mark D</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf3"/><xref ref-type="other" rid="dataro1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Biochemistry, La Trobe Institute for Molecular Science</institution>, <institution>La Trobe University</institution>, <addr-line><named-content content-type="city">Melbourne</named-content></addr-line>, <country>Australia</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>m.hulett@latrobe.edu.au</email> (MDH);</corresp><corresp id="cor2"><label>*</label>For correspondence: <email>m.kvansakul@latrobe.edu.au</email> (MK)</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>01</day><month>04</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e01808</elocation-id><history><date date-type="received"><day>31</day><month>10</month><year>2013</year></date><date date-type="accepted"><day>22</day><month>02</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Poon et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Poon 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="elife01808.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01808.001</object-id><p>Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP<sub>2</sub>). We determined the crystal structure of a NaD1:PIP<sub>2</sub> complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP<sub>2</sub> molecules through a unique ‘cationic grip’ configuration. Site-directed mutagenesis of NaD1 confirms that PIP<sub>2</sub>-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP<sub>2</sub> that permeabilizes cells via a novel membrane disrupting mechanism.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.001">http://dx.doi.org/10.7554/eLife.01808.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01808.002</object-id><title>eLife digest</title><p>It is often said that attack is the best form of defense; and the immune systems of plants and animals will often target the cell membranes of microbes and other pathogens in order to defend themselves. Disrupting the cell membrane causes essential contents to leak from the cell, and eventually, the cell will burst and die.</p><p>Most plants and animals produce small proteins called defensins that kill microbes by attacking their cell membranes. These defensins are thought to either destabilize the cell membrane by coating its outer surface or to insert themselves into the membrane to form open pores that allow vital biomolecules to leak out of the cell. However, the exact mechanism by which defensins attack microbial membranes is not understood.</p><p>In this study, Poon, Baxter, Lay et al. show that a defensin called NaD1—which was isolated from the ornamental tobacco <italic>Nicotiana alata</italic>—binds to a molecule from the cell membrane called phosphatidylinositol 4,5-bisphosphate, or PIP<sub>2</sub> for short. By working out the three-dimensional structure of this complex, Poon, Baxter, Lay et al. show that it contains 14 PIP<sub>2</sub> molecules and 14 NaD1 molecules in an arch-shaped structure and suggest that sequestering large numbers of PIP<sub>2</sub> molecules in this way destabilizes the cell membrane of the microbe.</p><p>These findings raise a number of questions: are there other small proteins that can destabilize cell membranes in a similar manner to defensins? Do the immune systems of other organisms also recognize molecules from microbial cell membranes to trigger this kind of counterattack? Furthermore, since defensins can also kill tumor cells, a better understanding of how they work might also lead to new treatments for cancer and other diseases in humans.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.002">http://dx.doi.org/10.7554/eLife.01808.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>protein structure</kwd><kwd>X-ray crystallography</kwd><kwd>antimicrobial peptide</kwd><kwd>phospholipids</kwd><kwd>fungus</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>human</kwd><kwd>other</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>National Health and Medical Research Council Career Development Fellowship</institution></institution-wrap></funding-source><award-id>637372</award-id><principal-award-recipient><name><surname>Kvansakul</surname><given-names>Marc</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>National Health and Medical Research Council Project Grant</institution></institution-wrap></funding-source><award-id>APP1007918</award-id><principal-award-recipient><name><surname>Kvansakul</surname><given-names>Marc</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Australian Research Council Discovery Project Grant</institution></institution-wrap></funding-source><award-id>DP120102694</award-id><principal-award-recipient><name><surname>van der Weerden</surname><given-names>Nicole L</given-names></name><name><surname>Anderson</surname><given-names>Marilyn A</given-names></name><name><surname>Hulett</surname><given-names>Mark D</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>Australian Research Council Fellowship</institution></institution-wrap></funding-source><award-id>FT130101349</award-id><principal-award-recipient><name><surname>Kvansakul</surname><given-names>Marc</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 novel innate defense mechanism of cell lysis involves the coordinated oligomerization of a defensin by interaction with the membrane lipid, phosphatidylinositol 4,5-bisphosphate.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Host defense peptides, which include cationic antimicrobial peptides (CAPs), are a group of innate immune molecules produced by essentially all plant and animal species that act as a first line of defense against microbial invasion. In common with most innate immunity peptides, they are relatively small (typically <100 amino acid residues), are predominantly cationic, and typically harbor a substantial number of hydrophobic amino acids (<xref ref-type="bibr" rid="bib18">Hancock and Lehrer, 1998</xref>; <xref ref-type="bibr" rid="bib5">Brogden, 2005</xref>; <xref ref-type="bibr" rid="bib32">Lai and Gallo, 2009</xref>). Although originally identified due to their potent activity against microbial pathogens, several CAPs also exhibit cytolytic activity against a range of mammalian tumor cells (<xref ref-type="bibr" rid="bib39">Lichtenstein et al., 1986</xref>; <xref ref-type="bibr" rid="bib9">Cruciani et al., 1991</xref>; <xref ref-type="bibr" rid="bib19">Hancock and Sahl, 2006</xref>; <xref ref-type="bibr" rid="bib55">Schweizer, 2009</xref>).</p><p>The defensins are a family of CAPs that are ubiquitously expressed in plants, animals, insects, and fungi that play an important role in innate immune defense against microbial threats (<xref ref-type="bibr" rid="bib5">Brogden, 2005</xref>; <xref ref-type="bibr" rid="bib36">Lay and Anderson, 2005</xref>; <xref ref-type="bibr" rid="bib19">Hancock and Sahl, 2006</xref>; <xref ref-type="bibr" rid="bib32">Lai and Gallo, 2009</xref>). The plant defensins belong to a large family of molecules that are highly variable in sequence but have a conserved structure. The sequence variability leads to several biological functions including antimicrobial activity, regulation of plant development, and pollen tube guidance (<xref ref-type="bibr" rid="bib6">Carvalho and Gomes, 2009</xref>; <xref ref-type="bibr" rid="bib10">De Coninck et al., 2013</xref>). Even those plant defensins that have been ascribed antifungal activity have large differences in sequence and are likely to act by different mechanisms (<xref ref-type="bibr" rid="bib66">van der Weerden and Anderson, 2013</xref>). The plant defensins are small (∼5 kDa, 45–54 amino acids), basic, cysteine-rich proteins that display a family-defining disulfide bond array (in a C<sub>I</sub>–C<sub>VIII</sub>, C<sub>II</sub>–C<sub>V</sub>, C<sub>III</sub>–C<sub>VI</sub>, and C<sub>IV</sub>–C<sub>VII</sub> configuration) known as the cysteine-stabilized αβ (CSαβ) motif. This motif consists of a triple-stranded antiparallel β-sheet, which is cross-braced via three disulfide bonds at the core of the molecule to an α-helix (in a βαββ arrangement). The fourth conserved disulfide bond further rigidifies the protein by linking together the N- and C-terminal regions of the molecule, effectively generating a highly stable pseudocyclic molecule (<xref ref-type="bibr" rid="bib26">Janssen et al., 2003</xref>; <xref ref-type="bibr" rid="bib35">Lay et al., 2003b</xref>, <xref ref-type="bibr" rid="bib37">2012</xref>; <xref ref-type="bibr" rid="bib36">Lay and Anderson, 2005</xref>). This CSαβ fold is also conserved in defensins found in other organisms, including insects and fungi (<xref ref-type="bibr" rid="bib36">Lay and Anderson, 2005</xref>).</p><p>NaD1, a plant defensin isolated from the flowers of the ornamental tobacco (<italic>Nicotiana alata</italic>), exhibits potent antifungal activity against pathogenic fungi, including <italic>Fusarium oxysporum, Botrytis cinerea, Aspergillus niger, Cryptococcus species</italic>, as well as the yeasts <italic>Saccharomyces cerevisiae</italic> and <italic>Candida albicans</italic> (<xref ref-type="bibr" rid="bib34">Lay et al., 2003a</xref>, <xref ref-type="bibr" rid="bib35">2003b</xref>, <xref ref-type="bibr" rid="bib37">2012</xref>; <xref ref-type="bibr" rid="bib68">van der Weerden et al., 2008</xref>, <xref ref-type="bibr" rid="bib67">2010</xref>; <xref ref-type="bibr" rid="bib20">Hayes et al., 2013</xref>). NaD1 inhibits fungal growth in a three-stage process that involves specific interaction with the cell wall and entry into the cytoplasm before cell death (<xref ref-type="bibr" rid="bib68">van der Weerden et al., 2008</xref>, <xref ref-type="bibr" rid="bib67">2010</xref>). Interaction with NaD1 also leads to hyper-production of reactive oxygen species, inducing oxidative damage that contributes to its fungicidal activity on <italic>Candida albicans</italic> (<xref ref-type="bibr" rid="bib20">Hayes et al., 2013</xref>).</p><p>Many CAPs have been postulated to act at the level of the plasma membrane of target cells. Suggested mechanisms of action for membrane permeabilization are based on the (1) carpet, (2) barrel-stave, and (3) toroidal-pore models (reviewed in <xref ref-type="bibr" rid="bib5">Brogden, 2005</xref>). In the carpet model, the CAPs act like classic detergents, accumulating and forming a carpet layer on the membrane outer surface, leading to local disintegration (including membrane micellization or fragmentation) upon reaching a critical concentration. Other CAPs are suggested to aggregate on the membrane surface before inserting into the bilayer forming a ‘barrel-stave’ pore where the hydrophobic peptide regions align with the lipid core and the hydrophilic peptide regions form the interior of the pore. Alternatively, in the toroidal pore model, the CAPs induce the lipid monolayers to bend continuously through the pore, with the polar peptide faces associating with the polar lipid head groups (<xref ref-type="bibr" rid="bib5">Brogden, 2005</xref>).</p><p>Although these models have been useful for describing potential mechanisms underlying the antimicrobial activity of various CAPs, it is not clear how well they represent the actual configuration of CAPs at the membrane. Furthermore, the oligomeric state of CAPs required for their activity based on the postulated models remains unknown. Indeed, it has long been hypothesized that the molecules could form proteinaceous pores and function through insertion into membranes (<xref ref-type="bibr" rid="bib5">Brogden, 2005</xref>). However, to date, the structural basis of CAP activity at the target membrane has not been defined. In addition to the uncertainty about the configuration of CAPs at the membrane, the role of ligands in modulating the recognition of target surfaces by CAPs remains unclear.</p><p>One class of ligands that has been linked to plant defensin antifungal activity are sphingolipids (<xref ref-type="bibr" rid="bib72">Wilmes et al., 2011</xref>), a key component of fungal cell walls and membranes. Plant defensins that bind sphingolipids include RsAFP2 from radish (binds glucosylceramide, GlcCer) (<xref ref-type="bibr" rid="bib64">Thomma et al., 2003</xref>; <xref ref-type="bibr" rid="bib62">Thevissen et al., 2004</xref>), DmAMP1 from dahlia (binds mannose-(inositol-phosphate)<sub>2</sub>-ceramide, M(IP)<sub>2</sub>C) (<xref ref-type="bibr" rid="bib60">Thevissen et al., 2000</xref>, <xref ref-type="bibr" rid="bib61">2003</xref>), as well as the pea defensin Psd1 (<xref ref-type="bibr" rid="bib16">Goncalves et al., 2012</xref>) and sugarcane defensin Sd5 (<xref ref-type="bibr" rid="bib11">de Paula et al., 2011</xref>) that both bind membranes enriched for specific glycosphingolipids. MsDef1, a defensin from <italic>Medicago sativa</italic>, has also been implicated in binding sphingolipids, as a mutant of the fungus <italic>Fusarium graminearum</italic> that is depleted in glucosylceramide, is highly resistant to MsDef1 (<xref ref-type="bibr" rid="bib48">Ramamoorthy et al., 2007</xref>).</p><p>In this report, we have identified the cellular phospholipid phosphatidylinositol 4,5-bisphosphate (PIP<sub>2</sub>) as a key ligand that is recognized during membrane permeabilization of fungal and mammalian plasma membranes. Using X-ray crystallography, we have defined the molecular interaction of NaD1 with PIP<sub>2</sub> and demonstrate that NaD1 forms oligomeric complexes with PIP<sub>2</sub>. Structure-guided mutagenesis revealed a critical arginine residue (R40) that is pivotal for NaD1:PIP<sub>2</sub> oligomer formation and that oligomerization is required for plasma membrane permeabilization. Engagement of PIP<sub>2</sub> is mediated by NaD1 dimers that form a distinctive PIP<sub>2</sub>-binding ‘cationic grip’ that interacts with the head groups of two PIP<sub>2</sub> molecules. Functional assays using NaD1 mutants reveal that the mechanism of membrane permeabilization by NaD1 is likely to be conserved between fungal and mammalian tumor cells. Together, these data lead to a new perspective on the role of ligand binding and oligomer formation of defensins during membrane permeabilization.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>NaD1 binds phospholipids including phosphatidylinositol 4,5-bisphosphate (PIP<sub>2</sub>)</title><p>To define the molecular basis of NaD1 target cell membrane permeabilization activity, we set out to identify potential ligands for NaD1. Membrane lipids represent an attractive target for NaD1; therefore, we investigated whether NaD1 interacts with cellular lipids using protein–lipid overlay assays based on lipid strips immobilized with 100 pmoles of various biologically active lipids (<xref ref-type="bibr" rid="bib47">Poon et al., 2010</xref>; <xref ref-type="bibr" rid="bib46">Patel et al., 2013</xref>). NaD1 specifically bound to certain phospholipids, including several phosphatidylinositol mono-/bis-/tri-phosphates, phosphatidylserine, phosphatidic acid, cardiolipin, and sulfatide (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Interestingly, NaD1 bound the functionally important plasma membrane phospholipid PIP<sub>2</sub> (<xref ref-type="fig" rid="fig1">Figure 1A</xref>) but did not bind to a panel of other membrane lipids or sphingolipids. To confirm that the ability of NaD1 to engage PIP<sub>2</sub> was not a result of immobilization on the lipid strip, we confirmed that NaD1 also bound PIP<sub>2</sub> in the context of a membrane bilayer using a liposome pull-down assay (<xref ref-type="fig" rid="fig1">Figure 1B</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.003</object-id><label>Figure 1.</label><caption><title>Interaction of NaD1 with lipids.</title><p>(<bold>A</bold>) Detection of NaD1 binding to cellular lipids by protein-lipid overlay assay. Blots are representative of at least two independent experiments for each strip. (<bold>B</bold>) Binding of NaD1 to PIP<sub>2</sub>-containing liposomes. NaD1 in <bold>A</bold> and <bold>B</bold> was detected using a rabbit anti-NaD1 antibody.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.003">http://dx.doi.org/10.7554/eLife.01808.003</ext-link></p></caption><graphic xlink:href="elife01808f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01808.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Relative binding of NaD1 to lipids.</title><p>Quantitation by densitometry of the relative binding of NaD1 to lipids (normalized to PtdIns(4,5)P<sub>2</sub> in <bold>A</bold> and <bold>B</bold>, sulfatide in <bold>C</bold>) on a (<bold>A</bold>) Membrane Lipid Strip, (<bold>B</bold>) PIP Strip, and (<bold>C</bold>) SphingoStrip. Data shown as mean ± SD (n = 2) for <bold>A</bold> and <bold>C</bold>, and mean ± SEM (n = 3) for <bold>B</bold>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.004">http://dx.doi.org/10.7554/eLife.01808.004</ext-link></p></caption><graphic xlink:href="elife01808fs001"/></fig></fig-group></p></sec><sec id="s2-2"><title>PIP<sub>2</sub> binding to NaD1 leads to the formation of an arch-shaped oligomer</title><p>To gain insight into the NaD1:PIP<sub>2</sub> interaction at the atomic level, we determined the crystal structure of NaD1 in complex with PIP<sub>2</sub>. The structure of monomeric NaD1 (<xref ref-type="bibr" rid="bib37">Lay et al., 2012</xref>) was used to solve the structure of a NaD1:PIP<sub>2</sub> complex by molecular replacement and refined to a resolution of 1.6 Å with values of R<sub>work</sub>/R<sub>free</sub> of 0.155/0.184 (<xref ref-type="table" rid="tbl1">Table 1</xref>). Upon PIP<sub>2</sub> binding, NaD1 forms an arch composed of 14 NaD1 molecules (<xref ref-type="fig" rid="fig2">Figure 2A</xref>), with a final arch diameter of 90 Å and a width of 35 Å. The asymmetric unit contains all 14 NaD1 molecules that form the final arch, with the symmetry of the arch being entirely non-crystallographic. Fourteen PIP<sub>2</sub> molecules are bound in an extended binding groove (<xref ref-type="fig" rid="fig2">Figure 2B</xref>) on the inside of the arch (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). The entire oligomeric complex is held together by a complex network of interactions, which include numerous NaD1:NaD1 (<xref ref-type="fig" rid="fig3">Figure 3A,B</xref>) and NaD1:PIP<sub>2</sub> interactions (<xref ref-type="fig" rid="fig3">Figure 3C,D</xref>). Notably, the arch-shaped oligomer displays a small degree of pitch, which although noticeable is not sufficient to allow the formation of an extended coil in the crystal (<xref ref-type="fig" rid="fig2">Figure 2B</xref>).<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.005</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.01808.005">http://dx.doi.org/10.7554/eLife.01808.005</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th/><th>NaD1:PIP<sub>2</sub> native</th></tr></thead><tbody><tr><td>Data collection</td><td/></tr><tr><td> Space group</td><td>C222<sub>1</sub></td></tr><tr><td> Cell dimensions</td><td/></tr><tr><td> <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td>79.64, 132.04, 153.01</td></tr><tr><td> α, β, γ (°)</td><td>90.00, 90.00, 90.00</td></tr><tr><td> Wavelength (Å)</td><td>0.9537</td></tr><tr><td> Resolution (Å)<xref ref-type="table-fn" rid="tblfn1">*</xref></td><td>40.84–1.6 (1.69–1.60)</td></tr><tr><td> <italic>R</italic><sub>sym</sub> or <italic>R</italic><sub>merge</sub><xref ref-type="table-fn" rid="tblfn1">*</xref></td><td>0.092 (0.617)</td></tr><tr><td> <italic>I</italic>/σ<italic>I</italic><xref ref-type="table-fn" rid="tblfn1">*</xref></td><td>11.6 (2.2)</td></tr><tr><td> Completeness (%)<xref ref-type="table-fn" rid="tblfn1">*</xref></td><td>99.7 (94.7)</td></tr><tr><td> Redundancy<xref ref-type="table-fn" rid="tblfn1">*</xref></td><td>6.7 (5.4)</td></tr><tr><td>Refinement</td><td/></tr><tr><td> Resolution (Å)</td><td>40.37–1.6</td></tr><tr><td> No. reflections</td><td>105745</td></tr><tr><td> <italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub></td><td>0.155/0.184</td></tr><tr><td> No. atoms</td><td/></tr><tr><td> Protein</td><td>10326</td></tr><tr><td> Ligand/ion</td><td>845</td></tr><tr><td> Water</td><td>816</td></tr><tr><td> <italic>B</italic>-factors</td><td/></tr><tr><td> Protein</td><td>21.5</td></tr><tr><td> Ligand/ion</td><td>28.9</td></tr><tr><td> Water</td><td>31.2</td></tr><tr><td> R.m.s. deviations</td><td/></tr><tr><td> Bond lengths (Å)</td><td>0.010</td></tr><tr><td> Bond angles (°)</td><td>1.651</td></tr></tbody></table><table-wrap-foot><fn id="tblfn1"><label>*</label><p>Values in parentheses are for highest resolution shell.</p></fn></table-wrap-foot></table-wrap><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.006</object-id><label>Figure 2.</label><caption><title>Crystal structure of the NaD1:PIP<sub>2</sub> complex.</title><p>(<bold>A</bold>) Two orthogonal views of a cartoon representation of the NaD1:PIP<sub>2</sub> oligomer comprising 14 NaD1 monomers (shown as ribbons) and 14 PIP<sub>2</sub> molecules (shown as green sticks). The surface of the NaD1 oligomer is shown in gray. (<bold>B</bold>) Surface representation of the NaD1 14-mer, displaying the extended binding groove on the inside of the arch. Coloring is by atom type (N in blue, O in red, S in yellow, and C in gray). For clarity the 14 bound PIP<sub>2</sub> molecules were omitted.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.006">http://dx.doi.org/10.7554/eLife.01808.006</ext-link></p></caption><graphic xlink:href="elife01808f002"/></fig><fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.007</object-id><label>Figure 3.</label><caption><title>Detailed view of the crystal structure of the NaD1:PIP<sub>2</sub> complex.</title><p>In all panels, hydrogen bonds and salt bridges are shown as black dotted lines. (<bold>A</bold>) View of the interface of two NaD1 monomers revealing the hydrogen bonding pattern, with monomer I shown in cyan and monomer II in magenta. Secondary structure elements are labeled in black. For clarity bound PIP<sub>2</sub> molecules are omitted. (<bold>B</bold>) Cartoon diagram of four molecules of NaD1 forming a dimer of dimers. (<bold>C</bold>) PIP<sub>2</sub> binding site on monomer I. Cartoon diagram of the PIP<sub>2</sub> binding site in monomer I on dimeric NaD1. (<bold>D</bold>) PIP<sub>2</sub> binding site on monomer II. Cartoon diagram of the PIP<sub>2</sub> binding site on monomer II on dimeric NaD1.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.007">http://dx.doi.org/10.7554/eLife.01808.007</ext-link></p></caption><graphic xlink:href="elife01808f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01808.008</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Cartoon of two NaD1 dimers with four bound PIP<sub>2</sub> molecules.</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.008">http://dx.doi.org/10.7554/eLife.01808.008</ext-link></p></caption><graphic xlink:href="elife01808fs002"/></fig><fig id="fig3s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01808.009</object-id><label>Figure 3—figure supplement 2.</label><caption><title>Simulated anneal omit map of a single PIP<sub>2</sub> molecule bound to an NaD1 dimer, contoured at 1σ.</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.009">http://dx.doi.org/10.7554/eLife.01808.009</ext-link></p></caption><graphic xlink:href="elife01808fs003"/></fig></fig-group></p></sec><sec id="s2-3"><title>The NaD1:PIP<sub>2</sub> oligomer contains two distinct NaD1:NaD1 interfaces</title><p>The observed NaD1:PIP<sub>2</sub> oligomer can be described as an assembly of seven NaD1 dimers, which comprise two distinct NaD1:NaD1 interfaces. The first interface is formed by an antiparallel alignment of the β1-strand from each of two NaD1 molecules (monomers I and II) and exhibits two-fold symmetry between the associated monomers (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). It comprises an average buried surface area of 430 Å<sup>2</sup> and is formed by a network of six hydrogen bonds involving R1, K4, E6, E27, K45, and C47. This dimeric arrangement leads to the formation of a ‘cationic grip’ (<xref ref-type="fig" rid="fig4">Figure 4A,B</xref>), which is able to accommodate two PIP<sub>2</sub> head groups simultaneously (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). A second interface is formed by the dimeric NaD1 (comprising monomers I and II) and adjacent NaD1 monomers III and IV (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). This interface is formed by hydrogen bonds involving N8 of monomer I, R1, E2, K17; D31 of monomer II, R1, K17; D31 of monomer III; and N8 of monomer IV, effectively forming a dimer of dimers (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). The interactions between two dimers are repeated seven times to allow formation of the observed 14-mer. The full 14-mer is thus constructed using two different interfaces.<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.010</object-id><label>Figure 4.</label><caption><title>The dimeric NaD1 ‘cationic grip‘ with two bound PIP<sub>2</sub> molecules.</title><p>(<bold>A</bold>) Surface view in two orientations of a NaD1 dimer (monomer I in cyan and monomer II in magenta) with two bound PIP<sub>2</sub> molecules (yellow and green). (<bold>B</bold>) The same as in <bold>A</bold> except that the surface shows a qualitative electrostatic representation (blue is positive, red in negative, and white is uncharged or hydrophobic). Figure generated using Pymol.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.010">http://dx.doi.org/10.7554/eLife.01808.010</ext-link></p></caption><graphic xlink:href="elife01808f004"/></fig></p></sec><sec id="s2-4"><title>PIP<sub>2</sub> is bound in an extended binding groove</title><p>In addition to NaD1:NaD1 interactions, oligomer formation requires the presence of PIP<sub>2</sub>. NaD1 binds PIP<sub>2</sub> primarily via a ‘cationic grip’ that is created by a NaD1 dimer, which results in the formation of a distinct binding site (<xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2</xref>) formed by K4 together with residues 33–40, which comprise a characteristic ‘KILRR’ motif (<xref ref-type="fig" rid="fig3">Figure 3C,D</xref>). PIP<sub>2</sub> forms a dense network of hydrogen bonds involving K4, H33, K36, I37, L38, and R40 of a single NaD1 monomer. In oligomeric NaD1:PIP<sub>2</sub>, a single PIP<sub>2</sub> binding site also contains interactions with neighboring NaD1 monomers (<xref ref-type="fig" rid="fig3">Figure 3C,D</xref>; <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). Bound PIP<sub>2</sub> forms additional hydrogen bonds with R40 from monomer II and K36 from monomer IV′, with the full PIP<sub>2</sub> binding site in the oligomer comprising contributions from three different NaD1 molecules (<xref ref-type="fig" rid="fig3">Figure 3C,D</xref>). Consequently, oligomer formation appears to be highly cooperative, with multiple interactions between adjacent NaD1 and PIP<sub>2</sub> molecules required to form the observed 14-mer (<xref ref-type="fig" rid="fig3">Figure 3</xref>).</p></sec><sec id="s2-5"><title>NaD1:PIP<sub>2</sub> oligomers form readily in solution</title><p>To confirm that oligomer formation is not a crystallization artifact, we treated mixtures of NaD1 and PIP<sub>2</sub> in aqueous solution with the crosslinker BS<sup>3</sup>, which resulted in covalent cross-linking of multiple NaD1 molecules that occurred only in the presence of PIP<sub>2</sub> (<xref ref-type="fig" rid="fig5">Figure 5A</xref>), whereas NaD1 on its own only formed a dimer as reported previously (<xref ref-type="bibr" rid="bib37">Lay et al., 2012</xref>).<fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.011</object-id><label>Figure 5.</label><caption><title>NaD1 forms oligomers with PIP<sub>2</sub>.</title><p>(<bold>A</bold>) Ability of NaD1 to form multimers in the presence of PIP<sub>2</sub> as determined by protein–protein cross-linking with BS<sup>3</sup> followed by SDS-PAGE and Coomassie Brilliant Blue staining. (<bold>B</bold>) TEM of NaD1:PIP<sub>2</sub> complexes. TEM micrographs of NaD1 alone, PIP<sub>2</sub> alone, or NaD1 in complex with PIP<sub>2</sub>. Data in <bold>A</bold> and <bold>B</bold> are representative of at least two independent experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.011">http://dx.doi.org/10.7554/eLife.01808.011</ext-link></p></caption><graphic xlink:href="elife01808f005"/></fig></p></sec><sec id="s2-6"><title>NaD1:PIP<sub>2</sub> forms fibrils</title><p>We next imaged NaD1:PIP<sub>2</sub> oligomers using transmission electron microscopy (TEM). Complexes of NaD1:PIP<sub>2</sub> (1:1.2 molar ratio) were applied to a carbon-coated copper grid and imaged. Strikingly, long string-like fibrillar structures were observed when both NaD1 and PIP<sub>2</sub> were present, whereas they were absent on grids bearing either NaD1 or PIP<sub>2</sub> in isolation (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). Although the NaD1:PIP<sub>2</sub> oligomer we observed by crystallography displays a subtle pitch, it is not sufficient to allow continuous addition or concatenation of 14-mers to form the fibrils observed by TEM, with the ends of two 14-mers running into each other. However, given that the crystal structure of the oligomer reveal an outer diameter of 90 Å, with a corresponding diameter of the fibrils under TEM of 10 nM, additional twisting of the 14-mer could allow for the formation of continuous coils with a diameter to match the fibrils observed under TEM.</p></sec><sec id="s2-7"><title>PIP<sub>2</sub> binding and oligomerization of NaD1 are critical for fungal cell killing</title><p>Based on our oligomeric NaD1:PIP<sub>2</sub> structure, we performed site-directed mutagenesis on NaD1 to confirm the role of proposed key amino acid residues in PIP<sub>2</sub> binding, oligomerization, and fungal cell killing. Examination of the PIP<sub>2</sub> binding pockets in the NaD1:PIP2 oligomer suggests that R40, which contacts two adjacent PIP<sub>2</sub> molecules simultaneously and interacts with the phosphate moiety at position 4, is critical for cooperative binding of PIP<sub>2</sub> and therefore formation of the NaD1:PIP<sub>2</sub> oligomer (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). Mutation of R40 should not lead to loss of PIP<sub>2</sub> binding, since PIP<sub>2</sub> would still form five hydrogen bonds and ionic interactions with NaD1 and should only impact oligomerization. In contrast, I37 contributes to PIP<sub>2</sub> binding but not oligomerization. We generated recombinant proteins of NaD1 (rNaD1) and NaD1 mutants (rNaD1(R40E) and rNaD1(I37F)) and confirmed their correct folding by CD spectroscopy (data not shown) and evaluated the ability of the mutant NaD1 to bind phospholipids, undergo PIP<sub>2</sub>-induced oligomerization, and kill the filamentous fungus <italic>F. oxysporum</italic> f. sp. vasinfectum.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.012</object-id><label>Figure 6.</label><caption><title>Multimerization of the NaD1:PIP<sub>2</sub> complex.</title><p>(<bold>A</bold>) Schematic representation of residues from neighboring NaD1 monomers involved in binding two PIP<sub>2</sub> molecules. Ability of rNaD1, rNaD1(R40E), and rNaD1(I37F) to (<bold>B</bold>) bind cellular lipids by protein-lipid overlay assay, (<bold>C</bold>) form multimers in the presence of PIP<sub>2</sub> as determined by protein–protein cross-linking with BS<sup>3</sup> followed by SDS-PAGE and Coomassie Brilliant Blue staining, and (<bold>D</bold>) to inhibit fungal cell growth. Error bars in <bold>D</bold> indicate SEM (n = 3). Data in <bold>B</bold>–<bold>D</bold> are representative of at least two independent experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.012">http://dx.doi.org/10.7554/eLife.01808.012</ext-link></p></caption><graphic xlink:href="elife01808f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01808.013</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Relative binding of rNaD1, rNaD1(I37F), and rNaD1(R40E) to lipids.</title><p>Quantitation by densitometry of the relative binding (normalized to PtdIns(4,5)P<sub>2</sub>) of (<bold>A</bold>) rNaD1, (<bold>B</bold>) rNaD1(I37F), and (<bold>C</bold>) rNaD1(R40E) to lipids on PIP Strips. Data shown as mean ± SD (n = 2) for <bold>A</bold> and <bold>B</bold>, and mean ± SEM (n = 3) for <bold>C</bold>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.013">http://dx.doi.org/10.7554/eLife.01808.013</ext-link></p></caption><graphic xlink:href="elife01808fs004"/></fig></fig-group></p><p>As predicted, mutation of R40 to glutamic acid led to a largely unchanged binding to PIP<sub>2</sub>, with the remaining five hydrogen bonds and ionic interactions formed between PIP<sub>2</sub> and rNaD1(R40E) compensating for the loss of two ionic interactions as well as the charge repulsion. However, it did result in reduced binding to PI(4)P (<xref ref-type="fig" rid="fig6">Figure 6B</xref>) and oligomerization (<xref ref-type="fig" rid="fig6">Figure 6C</xref>) that correlated with substantially reduced fungal cell killing (<xref ref-type="fig" rid="fig6">Figure 6D</xref>). It is important to note that although PIP<sub>2</sub> binding was maintained, the loss of interaction with the 4-phosphate moiety of PIP<sub>2</sub> results in loss of cooperative binding and therefore ablation of oligomerization, which is critically dependent on R40 forming ‘bridging’ interactions between two neighboring PIP<sub>2</sub> molecules. In contrast, mutation of I37 to phenylalanine had little effect on PIP<sub>2</sub> binding specificity, oligomerization, and fungal cell killing (<xref ref-type="fig" rid="fig6">Figure 6B–D</xref>). These data support our defined NaD1-PIP<sub>2</sub> structure and demonstrate that the coordinated oligomerization of NaD1 by interaction with PIP<sub>2</sub> is an important event during fungal cell killing.</p></sec><sec id="s2-8"><title>NaD1 permeabilizes the plasma membrane of mammalian tumor cells</title><p>Since PIP<sub>2</sub> is a critical component of mammalian plasma membranes, we investigated whether NaD1 also harbored permeabilization activity against mammalian cells. To this end, we performed a flow cytometry-based cell permeabilization assay on U937 monocytic lymphoma cells to measure uptake of the membrane-impermeable nucleic acid dye propidium iodide (PI). NaD1 permeabilized the plasma membrane of the U937 cells and induced a change in cell morphology in a concentration-dependent manner (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). Furthermore, rapid leakage of intracellular ATP from U937 cells was observed within the first 200 s following exposure to NaD1 (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>).<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.014</object-id><label>Figure 7.</label><caption><title>NaD1 kills mammalian tumor cells by membrane permeabilization.</title><p>(<bold>A</bold>) Forward scatter, side scatter, and PI uptake analysis of U937 cells treated with NaD1. (<bold>B</bold>) Binding of FITC-dextran and (<bold>C</bold>) LDH release by NaD1-treated U937 cells. Error bars in <bold>C</bold> indicate SEM (n = 3). Data in <bold>A</bold>–<bold>C</bold> are representative of at least two independent experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.014">http://dx.doi.org/10.7554/eLife.01808.014</ext-link></p></caption><graphic xlink:href="elife01808f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01808.015</object-id><label>Figure 7—figure supplement 1.</label><caption><title>NaD1 rapidly permeabilizes U937 cells.</title><p>Ability of NaD1 (0.5–20 μM) to mediate the release of ATP from U937 cells was examined using the ATP bioluminescence assay. The energy-dependency of the light-emitting luciferase-catalyzed oxidation of luciferin was used to indirectly measure the amount of ATP released by permeabilized cells over 30 min.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.015">http://dx.doi.org/10.7554/eLife.01808.015</ext-link></p></caption><graphic xlink:href="elife01808fs005"/></fig><fig id="fig7s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01808.016</object-id><label>Figure 7—figure supplement 2.</label><caption><title>Reduced and alkylated NaD1 (NaD1<sub>R&A</sub>) does not permeabilize U937 cells.</title><p>Ability of NaD1<sub>R&A</sub> to permeabilize U937 cells was investigated using the (<bold>A</bold>) PI uptake, (<bold>B</bold>) LDH release, and (<bold>C</bold>) ATP bioluminescence assays, with native NaD1 being included as a positive control. Error bars in <bold>A</bold> and <bold>B</bold> indicate SEM (n = 3). Data in <bold>A</bold>–<bold>C</bold> are representative of at least two independent experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.016">http://dx.doi.org/10.7554/eLife.01808.016</ext-link></p></caption><graphic xlink:href="elife01808fs006"/></fig><fig id="fig7s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01808.017</object-id><label>Figure 7—figure supplement 3.</label><caption><title>Tumor/transformed cells are more susceptible to killing by NaD1 than normal/primary cells.</title><p>MTT cell viability assays were performed on the indicated cell lines. The IC<sub>50</sub> values are shown as mean ± SD (n≥ 2).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.017">http://dx.doi.org/10.7554/eLife.01808.017</ext-link></p></caption><graphic xlink:href="elife01808fs007"/></fig></fig-group></p><p>NaD1-mediated lysis of U937 cells was confirmed by the uptake of FITC-dextran (up to 40 kDa) and the release of lactate dehydrogenase (140 kDa) into the supernatant after NaD1 treatment (<xref ref-type="fig" rid="fig7">Figure 7B,C</xref>). In contrast, reduced and alkylated NaD1 (NaD1<sub>R&A</sub>) showed no cytotoxic activities against U937 cells (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2</xref>), confirming the importance of the NaD1 tertiary structure for the ability to induce membrane permeabilization. It should be noted that NaD1 also permeabilized a diverse range of normal primary human cells and tumor cell lines (<xref ref-type="fig" rid="fig7s3">Figure 7—figure supplement 3</xref>), with the highest levels of activity exhibited against tumor cell lines. Collectively, these data suggest that, in addition to antifungal activity, NaD1 also exhibits antiproliferative properties against mammalian cells.</p><p>We then sought to examine changes in cell morphology upon NaD1 treatment. Live confocal laser scanning microscopy (CLSM) revealed rapid changes on the cell surface of NaD1-permeabilized tumor cells and showed the formation of large plasma membrane blebs, with adherent cells (HeLa and PC3) forming multiple blebs of different sizes (<xref ref-type="other" rid="video1">Video 1</xref>) and non-adherent cells (U937) forming typically one to two large blebs (<xref ref-type="other" rid="video2">Video 2</xref>; <xref ref-type="fig" rid="fig8">Figure 8A</xref>). Moreover, bleb size was frequently larger than the actual cell (diameter >20 μm) and did not retract over a period of 20 min (<xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1</xref>).<media content-type="glencoe play-in-place height-250 width-310" id="video1" mime-subtype="avi" mimetype="video" xlink:href="elife01808v001.avi"><object-id pub-id-type="doi">10.7554/eLife.01808.018</object-id><label>Video 1.</label><caption><title>NaD1 rapidly induces membrane blebbing and permeabilization of HeLa cells.</title><p>Live CLSM of PKH67-stained HeLa cells in the presence of PI. Cells were imaged over a period of 10 min (5 s/frame), with NaD1 (10 μM) being added to cells at 1 min.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.018">http://dx.doi.org/10.7554/eLife.01808.018</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video2" mime-subtype="avi" mimetype="video" xlink:href="elife01808v002.avi"><object-id pub-id-type="doi">10.7554/eLife.01808.019</object-id><label>Video 2.</label><caption><title>Formation of a single large membrane bleb on a U937 cell following NaD1 treatment.</title><p>Three-dimensional reconstruction of CLSM images of a NaD1-treated (10 μM) PKH67-/PI-stained U937 cell.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.019">http://dx.doi.org/10.7554/eLife.01808.019</ext-link></p></caption></media><fig-group><fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.020</object-id><label>Figure 8.</label><caption><title>NaD1 induces membrane blebbing of tumor cells.</title><p>(<bold>A</bold>) CLSM of PKH67-stained NaD1 (10 μM) permeabilized HeLa and U937 cells. (<bold>B</bold>) CLSM of U937 cells treated with NaD1 (20 μM) in the presence of PI and 4 kDa FITC-dextran. Arrows indicate entry of PI. It should be noted that in this experiment the detector gain on the helium–neon laser (red channel) was increased compared to that used in <bold>A</bold> to enable visualization of the cell lysis events. Scale bars represent 10 μm. Data in <bold>A</bold> and <bold>B</bold> are representative of at least two independent experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.020">http://dx.doi.org/10.7554/eLife.01808.020</ext-link></p></caption><graphic xlink:href="elife01808f008"/></fig><fig id="fig8s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01808.021</object-id><label>Figure 8—figure supplement 1.</label><caption><title>NaD1-induced membrane blebs do not retract once U937 cells are permeabilized.</title><p>Live confocal laser scanning microscopy (CLSM) of NaD1-treated U937 cells in the presence of PI. Cells were imaged over a period of 40 min, with NaD1 (10 μM) being added at 7 min.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.021">http://dx.doi.org/10.7554/eLife.01808.021</ext-link></p></caption><graphic xlink:href="elife01808fs008"/></fig><fig id="fig8s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01808.022</object-id><label>Figure 8—figure supplement 2.</label><caption><title>NaD1-mediated membrane permeabilization occurs at the blebs of PC3 cells.</title><p>Live CLSM of PC3 cells treated with NaD1 in the presence of PI and 4 kDa FITC-dextran. Cells were imaged over a period of 10 min (5 s/frame), with NaD1 (20 μM), and 4 kDa FITC-dextran (100 μg/ml) being added to cells at 30 s. It should be noted that individual PC3 cells show a variable rate of permeabilization by NaD1 reflecting a kinetic effect but eventually all cells are permeabilized under the experimental conditions.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.022">http://dx.doi.org/10.7554/eLife.01808.022</ext-link></p></caption><graphic xlink:href="elife01808fs009"/></fig></fig-group></p><p>In our CLSM studies, we noticed that NaD1-induced membrane blebbing typically coincided with PI uptake (<xref ref-type="other" rid="video1">Video 1</xref>). To determine whether membrane blebbing occurs prior to, during, or following membrane permeabilization, we treated U937 cells with NaD1 in the presence of PI and 4 kDa FITC-dextran to monitor the entry of these molecules into NaD1-sensitive cells (<xref ref-type="fig" rid="fig8">Figure 8B</xref>; <xref ref-type="other" rid="video3">Video 3</xref>). FITC-dextran and NaD1 were added at 00:35 min, with FITC-dextran being excluded from cells with an intact membrane. Bleb formation was first observed for the cell located at the center of the panel at 03:25 min, with PI staining appearing at a specific point at the edge of the bleb. From 03:25 to 04:15 min, PI staining was observed in the bleb and the cytoplasm with FITC-dextran also entering the cell from the bleb site. At 04:20 min, PI-stained molecules were ‘expelled’ out of the cell, possibly at the same region that PI first entered the bleb. Similar results were also observed for PC3 cells (<xref ref-type="fig" rid="fig8s2">Figure 8—figure supplement 2</xref>). These data suggest that (i) small molecules such as PI can enter the cell initially at a ‘weakened’ point at the membrane bleb, (ii) the bleb continues to enlarge while PI and 4 kDa FITC-dextran enters, and (iii) intracellular contents are released at the bleb site, representing cytolysis.<media content-type="glencoe play-in-place height-250 width-310" id="video3" mime-subtype="avi" mimetype="video" xlink:href="elife01808v003.avi"><object-id pub-id-type="doi">10.7554/eLife.01808.023</object-id><label>Video 3.</label><caption><title>NaD1-mediated membrane permeabilization occurs at the blebs of U937 cells.</title><p>Live CLSM of U937 cells treated with NaD1 in the presence of PI and 4 kDa FITC-dextran. Cells were imaged over a period of 10 min (5 s/frame), with NaD1 (20 μM) and 4 kDa FITC-dextran (100 μg/ml) being added to cells at 30 s.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.023">http://dx.doi.org/10.7554/eLife.01808.023</ext-link></p></caption></media></p></sec><sec id="s2-9"><title>NaD1 interacts with phosphoinositides in cellular membranes of tumor cells</title><p>We next determined the specific mechanism by which NaD1 permeabilizes mammalian cells. Firstly, we tested the binding of BODIPY-labeled NaD1 to U937 cells. BODIPY-NaD1 permeabilized U937 cells at a level comparable to unlabeled NaD1 and bound to both viable (7AAD-negative) and permeabilized (7AAD-positive) cells, with more BODIPY-NaD1 bound to membrane-damaged cells (<xref ref-type="fig" rid="fig9">Figure 9A</xref>). These data suggest that NaD1 can interact with U937 cells prior to membrane permeabilization and accumulates on/within NaD1-sensitive cells.<fig id="fig9" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.024</object-id><label>Figure 9.</label><caption><title>Subcellular localisation of BODIPY-NaD1 in tumor cells.</title><p>(<bold>A</bold>) Detection of BODIPY-NaD1 binding to viable and permeabilized U937 cells by flow cytometry. (<bold>B</bold>) CLSM of subcellular localization of BODIPY-NaD1 (10 μM) on permeabilized U937, PC3, and HeLa cells. Scale bars represent 10 μm. Data in <bold>A</bold> and <bold>B</bold> are representative of at least two independent experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.024">http://dx.doi.org/10.7554/eLife.01808.024</ext-link></p></caption><graphic xlink:href="elife01808f009"/></fig></p><p>We then determined the subcellular localization of BODIPY-NaD1 on permeabilized tumor cells. BODIPY-NaD1 accumulated at membrane bleb(s), in the cytoplasm and nucleolus and possibly at certain cytoplasmic organelles in U937, PC3, and HeLa cells (<xref ref-type="fig" rid="fig9">Figure 9B</xref>; <xref ref-type="other" rid="video4 video5">Videos 4 and 5</xref>). It is worth noting that the formation of large plasma membrane blebs has been reported previously in mammalian cells in which physically- or chemically-induced detachment of the plasma membrane from the actin cortex had occurred, including through enzymatic modification or sequestration of the inner membrane phospholipid, PIP<sub>2</sub> (<xref ref-type="bibr" rid="bib45">Niebuhr et al., 2002</xref>; <xref ref-type="bibr" rid="bib57">Sheetz et al., 2006</xref>; <xref ref-type="bibr" rid="bib29">Keller et al., 2009</xref>).<media content-type="glencoe play-in-place height-250 width-310" id="video4" mime-subtype="avi" mimetype="video" xlink:href="elife01808v004.avi"><object-id pub-id-type="doi">10.7554/eLife.01808.025</object-id><label>Video 4.</label><caption><title>BODIPY-NaD1 accumulates at the plasma membrane and certain intracellular organelles.</title><p>Three-dimensional reconstruction of CLSM images of BODIPY-NaD1-treated (10 μM) PI-stained PC3 cells.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.025">http://dx.doi.org/10.7554/eLife.01808.025</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video5" mime-subtype="avi" mimetype="video" xlink:href="elife01808v005.avi"><object-id pub-id-type="doi">10.7554/eLife.01808.026</object-id><label>Video 5.</label><caption><title>BODIPY-NaD1 accumulates at the plasma membrane and certain intracellular organelles.</title><p>Three-dimensional reconstruction of CLSM images of BODIPY-NaD1-treated (10 μM) PI-stained HeLa cells.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.026">http://dx.doi.org/10.7554/eLife.01808.026</ext-link></p></caption></media></p><p>We then asked whether the binding of NaD1 to PIP<sub>2</sub> at the inner leaflet of the plasma membrane could lead to the formation of blebs, as PIP<sub>2</sub> is a key mediator of cytoskeleton-membrane interactions (<xref ref-type="bibr" rid="bib50">Raucher et al., 2000</xref>; <xref ref-type="bibr" rid="bib56">Sheetz, 2001</xref>). HeLa cells expressing GFP-PH(PLCδ), which binds specifically to PIP<sub>2</sub>, were treated with NaD1 and showed a marked delay from the initiation of blebbing (rapid small membrane blebbing) to membrane permeabilization compared with cells expressing free GFP (<xref ref-type="fig" rid="fig10">Figure 10</xref>; <xref ref-type="other" rid="video6 video7">Videos 6 and 7</xref>). Quantitation of the kinetics of cell permeabilization indicated that GFP-PH(PLCδ)-expressing cells took approximately 2.5 times as long as GFP only cells to permeabilize in response to NaD1 (235 ± 40 s vs 90 ± 20 s, respectively) (<xref ref-type="fig" rid="fig10">Figure 10B</xref>). These results suggest that the expression of GFP-PH(PLCδ) may compete with NaD1 for PIP<sub>2</sub> binding at the inner leaflet of the plasma membrane and interfere with NaD1-induced cell permeabilization.<fig id="fig10" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.027</object-id><label>Figure 10.</label><caption><title>Expression of GFP-PH(PLCδ) in HeLa cells significantly delays NaD1-mediated cell permeabilization compared with cells expressing free GFP.</title><p>(<bold>A</bold>) CLSM of NaD1 (10 μM) treated HeLa cells expressing GFP-PH(PLCδ). Scale bars represent 10 μm. (<bold>B</bold>) The average length of time taken for NaD1 (10 μM) to permeabilize (PI-positive) GFP-PH(PLCδ)-expressing vs free GFP-expressing HeLa cells were analyzed over a period of 15 min. For GFP-PH(PLCδ)-expressing cells, n = 21; for free GFP-expressing cells, n = 29. Error bars indicate SEM, * = p<0.005.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.027">http://dx.doi.org/10.7554/eLife.01808.027</ext-link></p></caption><graphic xlink:href="elife01808f010"/></fig><media content-type="glencoe play-in-place height-250 width-310" id="video6" mime-subtype="avi" mimetype="video" xlink:href="elife01808v006.avi"><object-id pub-id-type="doi">10.7554/eLife.01808.028</object-id><label>Video 6.</label><caption><title>Expression of GFP-PH(PLCδ) in HeLa cells delays NaD1-mediated cell permeabilization.</title><p>Live CLSM of GFP-PH(PLCδ) transfected HeLa cells treated with NaD1 in the presence of PI. Cells were imaged over a period of 15 min (5 s/frame), with NaD1 (10 μM) being added to cells at 30 s.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.028">http://dx.doi.org/10.7554/eLife.01808.028</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video7" mime-subtype="avi" mimetype="video" xlink:href="elife01808v007.avi"><object-id pub-id-type="doi">10.7554/eLife.01808.029</object-id><label>Video 7.</label><caption><title>Expression of free GFP in HeLa cells does not delay NaD1-mediated cell permeabilization.</title><p>Live CLSM of free GFP transfected HeLa cells treated with NaD1 in the presence of PI. Cells were imaged over a period of 20 min (5 s/frame), with NaD1 (10 μM) being added to cells at 30 s.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.029">http://dx.doi.org/10.7554/eLife.01808.029</ext-link></p></caption></media></p><p>We then evaluated the effect of our loss-of-function mutant rNaD1(R40E), which exhibited reduced killing activity of fungal cells, on U937 cells. rNaD1(R40E) showed dramatically reduced ability to permeabilize these tumor cells, in contrast to the control rNaD1 and rNaD1(I37F) proteins as demonstrated by the PI uptake assay (<xref ref-type="fig" rid="fig11">Figure 11A</xref>). Similarly, rNaD1(R40E)-treated U937 cells displayed no LDH release (<xref ref-type="fig" rid="fig11">Figure 11B</xref>) or FITC-dextran uptake (<xref ref-type="fig" rid="fig11">Figure 11C</xref>) compared to the control proteins.<fig id="fig11" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.030</object-id><label>Figure 11.</label><caption><title>Permeabilization of U937 cells is impaired in rNaD1(R40E).</title><p>Ability of rNaD1, rNaD1(R40E) and rNaD1(I37F) to permeabilize U937 cells as assessed by (<bold>A</bold>) PI uptake, (<bold>B</bold>) LDH release, and (<bold>C</bold>) FITC-dextran binding assays. Error bars in <bold>A</bold> and <bold>B</bold> indicate SEM (n = 3). Data in <bold>A</bold>–<bold>C</bold> are representative of at least two independent experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.030">http://dx.doi.org/10.7554/eLife.01808.030</ext-link></p></caption><graphic xlink:href="elife01808f011"/></fig></p><p>In their totality, these data support our notion that the coordinated oligomerization of NaD1 by interaction with PIP<sub>2</sub> is a critical event for fungal and tumor cell killing.</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>CAPs, of which defensins are a major family, are weapons within the armory of host defense peptides that are utilized by animals and plants in their fight against pathogenic threats. NaD1 is a defensin from the ornamental tobacco that has potent activity against fungi and yeast (<xref ref-type="bibr" rid="bib34">Lay et al., 2003a</xref>, <xref ref-type="bibr" rid="bib37">2012</xref>; <xref ref-type="bibr" rid="bib68">van der Weerden et al., 2008</xref>, <xref ref-type="bibr" rid="bib67">2010</xref>; <xref ref-type="bibr" rid="bib20">Hayes et al., 2013</xref>). This defensin operates by a three-step mechanism—specific interaction with the cell wall followed by permeabilization of the plasma membrane and entry into the cytoplasm (<xref ref-type="bibr" rid="bib67">van der Weerden et al., 2010</xref>). However, the precise molecular basis of membrane permeabilization and passage through the membrane is poorly defined, particularly in terms of the specific lipid targets on the membrane and the structural definition of defensin-lipid interactions.</p><p>In this study, we identified the lipid targets of NaD1 as phosphoinositides (PIPs) and show that the binding of particular PIPs such as PIP<sub>2</sub>, mediates NaD1 oligomerization and membrane permeabilization. This expands on our previous study revealing that the ability of NaD1 to homo-dimerize enhances its antifungal activity (<xref ref-type="bibr" rid="bib37">Lay et al., 2012</xref>). It has been postulated that the ability of human defensins and other CAPs to form higher oligomeric states at the plasma membranes of target cells is a contributing factor in membrane disruption and/or permeabilization (<xref ref-type="bibr" rid="bib22">Hill et al., 1991</xref>; <xref ref-type="bibr" rid="bib73">Wimley et al., 1994</xref>; <xref ref-type="bibr" rid="bib23">Hoover et al., 2000</xref>; <xref ref-type="bibr" rid="bib42">Mader and Hoskin, 2006</xref>; <xref ref-type="bibr" rid="bib70">Wei et al., 2010</xref>), but to date no structural explanations have been reported. Our crystal structure of a NaD1:PIP<sub>2</sub> oligomeric complex reveals the first detailed molecular description of a plant defensin-lipid interaction and identifies a new mechanism of membrane permeabilization.</p><p>The proposed antifungal mechanisms of action for plant defensins are diverse and includes membrane permeabilization, generation of reactive oxygen species with induction of apoptosis, and dysregulation of Ca<sup>2+</sup> influx and K<sup>+</sup> efflux (reviewed in <xref ref-type="bibr" rid="bib10">De Coninck et al., 2013</xref>; <xref ref-type="bibr" rid="bib66">van der Weerden and Anderson, 2013</xref>). The structural basis of the interaction of these defensins with lipid has not been defined. However, it is interesting to note that the equivalent region to the ‘KILRR’ loop of NaD1 (the loop between the β2 and β3 strands), that is critical in forming the lipid-binding ‘cationic grip’, has been implicated as functionally important for the antifungal activity of a number of plant defensins. For example, mutations in the equivalent region of RsAFP2 abolished antifungal activity (<xref ref-type="bibr" rid="bib12">De Samblanx et al., 1997</xref>), and a chimeric protein generated by replacing this region of MsDef1 with the equivalent region of MtDef4 (a functionally distinct defensin that does not bind sphingolipid) resulted in functional conversion into a defensin able to inhibit the growth of a glucosylceramide-deficient, MsDef1-resistant <italic>F. graminearum</italic> strain (<xref ref-type="bibr" rid="bib52">Sagaram et al., 2011</xref>). Based on our observation that NaD1 binds phosphoinositides through the same loop region, it is tempting to speculate that plants have evolved a suite of defensins that specifically interact through their β2–β3 loop regions with different membrane lipids to mediate fungal cell killing. The β2–β3 region has also been implicated in the α-amylase activity of the plant defensin, VrD1, suggesting that this region is also functionally important in defensins with different activities (<xref ref-type="bibr" rid="bib41">Lin et al., 2007</xref>). Not surprisingly, the β2–β3 region of plant defensins exhibits considerable sequence divergence which is likely to reflect their ability to bind different ligands and therefore the various mechanisms of antifungal activity (<xref ref-type="bibr" rid="bib66">van der Weerden and Anderson, 2013</xref>).</p><p>The antifungal plant defensins, DmAMP1 and RsAFP2, interact with different sphingolipids. DmAMP1 binds M(IP)<sub>2</sub>C (<xref ref-type="bibr" rid="bib60">Thevissen et al., 2000</xref>, <xref ref-type="bibr" rid="bib61">2003</xref>) and <italic>S. cerevisiae</italic> strains that have gene disruptions to encoded proteins within the M(IP)<sub>2</sub>C biosynthetic pathway are rendered DmAMP1-resistant (<xref ref-type="bibr" rid="bib63">Thevissen et al., 2005</xref>). In contrast, RsAFP2 binds to GlcCer, present in fungi such as <italic>Pichia pastoris</italic> and <italic>C. albicans</italic> and does not cause permeabilization or growth inhibition of strains that do not express GlcCer (<xref ref-type="bibr" rid="bib62">Thevissen et al., 2004</xref>). The fact that DmAMP1 is able to act on these strains supports the notion that DmAMP1 and RsAFP2 have different lipid targets. It is worthwhile noting that RsAFP2 does not bind to GlcCer derived from human or soybean (<xref ref-type="bibr" rid="bib62">Thevissen et al., 2004</xref>), suggesting that there is some level of selectivity, while its inability to permeabilize artificial liposomes containing GlcCer indicates that binding alone is insufficient for its permeabilization action (<xref ref-type="bibr" rid="bib62">Thevissen et al., 2004</xref>).</p><p>This is the first report of a defensin from any species targeting a phosphoinositide, such as PIP<sub>2</sub>. Interestingly, a number of toxins have been reported to directly or indirectly target PIP<sub>2</sub>, resulting in plasma membrane reorganization and permeabilization. The marine bacterium <italic>Vibrio parahaemolyticus</italic> causes gastroenteritis in humans by acting as an inositol polyphospholipid 5′ phosphatase. It targets PIP<sub>2</sub> by catalyzing the removal of the 5′ phosphate moiety, resulting in the disruption of PIP<sub>2</sub>-mediated cytoskeletal interactions leading to membrane blebbing and cell lysis (<xref ref-type="bibr" rid="bib4">Broberg et al., 2010</xref>). A similar mode of action has also been described for the effector protein ipgD of the bacillary dysentery causing Gram-negative pathogen <italic>Shigella flexneri</italic> (<xref ref-type="bibr" rid="bib45">Niebuhr et al., 2002</xref>). The equinatoxin from the sea anemone <italic>Actinia equina</italic> also causes membrane blebbing and cell lysis via a mechanism involving Ca<sup>2+</sup>-mediated PIP<sub>2</sub> hydrolysis at the inner membrane and the formation of membrane pores in target cells (<xref ref-type="bibr" rid="bib15">Garcia-Saez et al., 2011</xref>). In this study, we show that NaD1 also causes plasma membrane blebbing and permeabilization of human cells. However, in contrast to the enzymatic or pore-forming mechanism(s) of the above toxins, it does so through the direct binding of PIP<sub>2</sub>. The ability of NaD1 to form an oligomeric complex with PIP<sub>2</sub> suggests it could potentially sequester PIP<sub>2</sub> from the plasma membrane, leading to membrane destabilization, blebbing, and ultimately cell lysis. Such an oligomerization process would be predicted to efficiently displace any PIP<sub>2</sub>-binding molecules. Indeed, the ability of lipid-binding proteins to oligomerize on membranes has been proposed as an important mechanism in mediating high-avidity membrane interactions (<xref ref-type="bibr" rid="bib38">Lemmon, 2008</xref>).</p><p>The increased sensitivity of tumor cells to NaD1 compared with its effects on healthy primary cells may be attributed to a number of differences in the physical properties of the plasma membranes of these two cell types. These include an increase in the expression of negatively charged outer membrane components, such as O-glycosylated mucins (<xref ref-type="bibr" rid="bib74">Yoon et al., 1996</xref>; <xref ref-type="bibr" rid="bib30">Kufe, 2009</xref>) and phosphatidylserine (<xref ref-type="bibr" rid="bib65">Utsugi et al., 1991</xref>; <xref ref-type="bibr" rid="bib49">Ran et al., 2002</xref>), which could allow stronger initial electrostatic interactions between the cationic NaD1 and the cell surface. The increased levels of microvilli (<xref ref-type="bibr" rid="bib7">Chaudhary and Munshi, 1995</xref>; <xref ref-type="bibr" rid="bib25">Ino et al., 2002</xref>) and higher degree of membrane fluidity (<xref ref-type="bibr" rid="bib58">Sok et al., 1999</xref>; <xref ref-type="bibr" rid="bib75">Zeisig et al., 2007</xref>) in tumor cells may also facilitate the aggregation of a greater number of NaD1 molecules to the cell surface and assist in penetration and/or destabilization of the membrane, respectively. The precise mechanism of action for the increased sensitivity of mammalian tumor cells over normal cells to defensins and whether this approach can be harnessed for selective tumor cell killing remains to be determined.</p><p>The ability of NaD1 to permeabilize both fungal and mammalian cell membranes suggests a common mode of interaction with membranes and our findings described herein implicate PIP<sub>2</sub> in both settings. Although the plasma membranes on mammalian and fungal cells are different in overall lipid compositions (typically mammalian cells are rich in zwitterionic phospholipids whereas fungal membrane have higher levels of anionic phospholipids), PIP<sub>2</sub> is important in both species (<xref ref-type="bibr" rid="bib13">Di Paolo and De Camilli, 2006</xref>; <xref ref-type="bibr" rid="bib69">van Meer et al., 2008</xref>). PIP<sub>2</sub> is normally found on the inner leaflet of mammalian cells and although it is a minor species comprising only 0.5–1% of phospholipids, it plays a major regulatory role in a number of important membrane-related processes, including signal transduction, ion channel function, and cytoskeletal attachment (<xref ref-type="bibr" rid="bib43">McLaughlin and Murray, 2005</xref>). Similar functions for PIP2 have been reported or suggested in the plasma membrane of fungi (<xref ref-type="bibr" rid="bib69">van Meer et al., 2008</xref>). The importance of PIP2 in many fundamental cellular processes would certainly make it an attractive target for defense against pathogens.</p><p>In addition to binding PIP<sub>2</sub>, we show that NaD1 is also able to bind to a number of other phospholipids. The functional consequences of the promiscuous binding by NaD1 remains to be determined. The various phospholipids recognized by NaD1 have different subcellular distributions and functions. In its normal physiological setting as a plant antifungal molecule, the ability of NaD1 to bind a number of different phospholipids may enable defense against a wide array of different fungal pathogens. How NaD1 is able to enter cells remains to be defined. It is possible that the binding of PIPs such as PI(3)P may mediate entry of NaD1 into cells. Indeed, cell surface-expressed PI(3)P has been suggested to mediate entry of eukaryotic pathogen effector molecules, such as Avr from oomycetes and fungi, into plant cells (<xref ref-type="bibr" rid="bib28">Kale et al., 2010</xref>).</p><p>Structurally, one can envisage that PIPs other than PIP<sub>2</sub> can fit into the cationic grip and be able to induce NaD1 oligomerization. Inspection of the cationic grip indicates that an additional phosphate group at the 3-inositol position can be accommodated and would contact R40 in a manner similar to the phosphate in the 4-inositol position (<xref ref-type="fig" rid="fig12">Figure 12</xref>) and may induce oligomerization in a manner analogous to the 4-phosphate. In contrast, a phosphate at the 6-inositol position is unlikely to be tolerated without reorganization of the binding site due to a steric clash with S35. Overall, a combination of phosphate groups on the inositol ring in position 5, together with an additional phosphate in position 3 or 4, appears to be capable of oligomerization. Furthermore, PIPs harboring phosphate groups in positions 3, 4, and 5 should be well tolerated in the grip.<fig id="fig12" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.031</object-id><label>Figure 12.</label><caption><title>PIP<sub>2</sub> fit into the NaD1 ‘cationic grip’.</title><p>Cut-away of PIP<sub>2</sub> bound in the NaD1 cationic grip. NaD1 dimer surface is shown in gray, PIP<sub>2</sub> in green and select NaD1 residues are shown as stick representation. NaD1 monomeric chains are colored in gray and salmon. Carbon atoms in the inositol ring are numbered (1–6).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.031">http://dx.doi.org/10.7554/eLife.01808.031</ext-link></p></caption><graphic xlink:href="elife01808f012"/></fig></p><p>Our structure of the NaD1:PIP<sub>2</sub> complex suggests that formation of the cationic grip via a NaD1 dimer is important for the formation of a functional PIP<sub>2</sub> binding site. Notably, this dimeric arrangement is different to the dimers we previously observed for NaD1 alone (<xref ref-type="bibr" rid="bib37">Lay et al., 2012</xref>). Although one of the two dimer configurations observed was based on an interface formed by two opposing β1 strands, the overall configuration places the β2–β3 loops at opposite ends of the dimer (<xref ref-type="bibr" rid="bib37">Lay et al., 2012</xref>), so that the ‘cationic grip’ is absent. Consequently, a substantial reorientation is required to convert the NaD1 dimer observed in the absence of PIP<sub>2</sub> into a configuration that can engage PIP<sub>2</sub> and oligomerize.</p><p>Although CAP:lipid interactions (e.g., nisin:lipid II, plectasin:lipid II) have been investigated using structural methods (<xref ref-type="bibr" rid="bib24">Hsu et al., 2004</xref>; <xref ref-type="bibr" rid="bib54">Schneider et al., 2010</xref>), the formation of defensin:ligand oligomers as observed for NaD1:PIP<sub>2</sub> is unique. NaD1 engages PIP<sub>2</sub> in a cooperative manner where three NaD1 monomers form a complete PIP<sub>2</sub> binding site, with each NaD1 monomer participating in the formation of three distinct PIP<sub>2</sub> binding sites. This ability to participate in the engagement of multiple PIP<sub>2</sub> molecules is mediated by two key basic residues, K4 and R40. The lantibiotic peptide nisin binds specifically to the membrane-bound cell wall precursor lipid II [undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc], leading to pore formation and permeabilization of the bacterial cytoplasmic membrane (<xref ref-type="bibr" rid="bib53">Sahl and Bierbaum, 1998</xref>; <xref ref-type="bibr" rid="bib71">Wiedemann et al., 2001</xref>). Structural studies have revealed that nisin forms a 1:1 complex with lipid II (<xref ref-type="bibr" rid="bib24">Hsu et al., 2004</xref>), however the precise contribution of lipid II binding to plasma membrane permeabilization is not fully established. Interestingly, the report on the nisin:lipid II structure indicated that nisin precipitated upon lipid II addition and the structure could only be determined by dissolving the nisin:lipid II complex in DMSO (<xref ref-type="bibr" rid="bib24">Hsu et al., 2004</xref>). It is conceivable that nisin also forms oligomers that were present in the precipitated sample that was discarded. The fungal defensin plectasin is another antimicrobial peptide that requires lipid II binding for its biological activity (<xref ref-type="bibr" rid="bib54">Schneider et al., 2010</xref>). NMR spectroscopy was used to map a putative lipid II binding site on plectasin, however the interaction appears again to be a 1:1 complex, with no suggestion of oligomer formation. Furthermore, plectasin does not permeabilize target microbial membranes and acts by directly targeting bacterial cell wall biosynthesis (<xref ref-type="bibr" rid="bib54">Schneider et al., 2010</xref>).</p><p>Amongst the many described phospholipid-binding proteins, a number of different domain structures have been defined that exhibit stereospecific recognition of specific phosphoinositide head groups in the context of cellular membrane surfaces. These include the pleckstrin homology (PH), ‘Fab1, YOTB, Vac1, EEA1’ (FYVE), and Phox-homology (PX) domains (<xref ref-type="bibr" rid="bib3">Bravo et al., 2001</xref>; <xref ref-type="bibr" rid="bib38">Lemmon, 2008</xref>), with all three showing structural similarities to that we describe for PIP<sub>2</sub> binding by NaD1. In each of these domains, phosphoinositide binding is mediated through pockets that are strategically lined with basic residues formed by two distal β loops. Pleckstrin homology domains consist of two perpendicular antiparallel β-sheets followed by a C-terminal amphipathic helix, with the canonical phosphoinositide binding pocket comprising basic residues derived from two distal β-loops on the two β-sheets (<xref ref-type="bibr" rid="bib40">Lietzke et al., 2000</xref>). PX domains contain a triple-stranded antiparallel β-sheet followed by a helical subdomain made up of four α-helices, with the β1 and β2 loops together with α-helix 3 forming a positively-charged pocket responsible for binding phosphoinositides (<xref ref-type="bibr" rid="bib3">Bravo et al., 2001</xref>). FYVE domains are small cysteine-rich Zn<sup>2+</sup> binding domains, consisting of two β-strands followed by a small C-terminal α-helix, with the binding pocket for the inositol head group of PI(3)P comprising basic clusters at either end of the β1 strand, one of which possesses a common (R/K)(R/K)HHCR motif (<xref ref-type="bibr" rid="bib31">Kutateladze and Overduin, 2001</xref>). The topology of the phosphoinositide binding pocket formed by the NaD1 dimer is similar to that of the PH domains, however the formation of the pocket is unique and involves the symmetrical juxtaposition of identical β2–β3 loops (comprised of KILRR) by dimerization of two NaD1 monomers. Thus, despite the very different overall folds of NaD1 and the other phosphoinositide binding domains, key structural features that govern how they recognize phosphoinositides are generally conserved.</p><p>It is of interest that a recent report describes the ability of human α-defensin 6 to self-assemble into high-order oligomers termed nanonets (<xref ref-type="bibr" rid="bib8">Chu et al., 2012</xref>). In contrast to NaD1:PIP<sub>2</sub> oligomers, these fibril-like structures appear to form without involvement of defined extrinsic ligands and rely on stochastic binding to bacterial surface proteins to initiate self-assembly. Although composed of defensins, these nanonets do not harbor direct antibacterial activity per se, but rather act by trapping bacteria to prevent cellular adhesion and invasion (<xref ref-type="bibr" rid="bib8">Chu et al., 2012</xref>). Together with our findings, these studies indicate that defensins are able to form different fibrils or oligomers for diverse functions in innate immunity.</p><p>Certain CAPs and cell penetrating peptides have been reported to penetrate biological membranes with or without membrane permeabilization (<xref ref-type="bibr" rid="bib21">Henriques et al., 2006</xref>; <xref ref-type="bibr" rid="bib2">Ashida et al., 2011</xref>). Although it is yet to be elucidated how NaD1 could pass through the plasma membrane to interact with phospholipids at the inner leaflet, our data provide several significant insights. NaD1 permeabilizes mammalian cells by forming a novel phosphoinositide recognition complex associated with the formation of membrane blebs and membrane rupture, possibly involving disruption of cytoskeleton-membrane interactions through the binding of PIP<sub>2</sub> at the inner leaflet (<xref ref-type="fig" rid="fig13">Figure 13</xref>). This novel mechanism of cell lysis is distinct from that proposed for other CAPs that act via pore formation or a non-specific charge-based interaction with the plasma membrane (<xref ref-type="bibr" rid="bib5">Brogden, 2005</xref>) and the well-defined pore-forming ability of cholesterol-dependent cytolysins (<xref ref-type="bibr" rid="bib51">Rossjohn et al., 1997</xref>), perforin (<xref ref-type="bibr" rid="bib33">Law et al., 2010</xref>) or complement membrane attack complex (<xref ref-type="bibr" rid="bib17">Hadders et al., 2007</xref>). These findings not only reveal a new mechanism of cell lysis but also uncover a potential evolutionarily conserved innate defense mechanism that can target cell membranes through the recognition of a ‘phospholipid pattern/code’.<fig id="fig13" position="float"><object-id pub-id-type="doi">10.7554/eLife.01808.032</object-id><label>Figure 13.</label><caption><title>Proposed molecular mechanism of NaD1-mediated tumor cell lysis.</title><p>Schematic representation of NaD1-induced membrane blebbing and permeabilization. The assembly of NaD1:PIP<sub>2</sub> oligomer can potentially be formed by (i) sequential recruitment of a NaD1 monomer followed by a PIP<sub>2</sub> molecule or (ii) dimerization of two single NaD1:PIP<sub>2</sub> complex followed by the recruitment of NaD1:PIP<sub>2</sub> dimers.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01808.032">http://dx.doi.org/10.7554/eLife.01808.032</ext-link></p></caption><graphic xlink:href="elife01808f013"/></fig></p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Purification of NaD1</title><p>NaD1 was isolated from its natural source, whole <italic>Nicotiana alata</italic> flowers, as described previously (<xref ref-type="bibr" rid="bib68">van der Weerden et al., 2008</xref>).</p></sec><sec id="s4-2"><title>Preparation of reduced and alkylated NaD1 (NaD1<sub>R&A</sub>)</title><p>Reduction and alkylation of NaD1 was performed as described previously (<xref ref-type="bibr" rid="bib68">van der Weerden et al., 2008</xref>).</p></sec><sec id="s4-3"><title>BODIPY labeling of NaD1</title><p>BODIPY labeling of NaD1 was performed as described previously (<xref ref-type="bibr" rid="bib67">van der Weerden et al., 2010</xref>).</p></sec><sec id="s4-4"><title>Protein-lipid overlay assay</title><p>NaD1 binding to lipids spotted on Membrane Lipid Strips, PIP Strips or SphingoStrips was performed according to manufacturer’s instructions (Echelon Biosciences, Salt Lake City, UT). Briefly, lipid strips were incubated with PBS/3% fatty acid-free BSA for 120 min at RT to block non-specific binding. The lipid strips were then incubated with NaD1 or NaD1 mutants (1 μg/ml) diluted in PBS/1% fatty acid-free BSA for 60 min at 4°C and washed thoroughly for 60 min at RT with PBS/0.1% Tween-20. Membrane-bound NaD1 was detected by probing the lipid strips with 2 μg/ml of a protein A purified rabbit anti-NaD1 antibody (<xref ref-type="bibr" rid="bib68">van der Weerden et al., 2008</xref>) diluted in PBS/1% fatty acid-free BSA for 60 min at 4°C, followed by a HRP-conjugated donkey anti-rabbit Ig antibody (GE Healthcare, Buckinghamshire, United Kingdom) diluted to 1:2000 in PBS/1% fatty acid-free BSA for 60 min at 4°C. After each antibody incubation, the lipid strips were washed for 30 min at RT with PBS/0.1% Tween-20. Chemiluminescence was detected using the enhanced chemiluminescence reagent (GE Healthcare) and developed using Hyperfilm (GE Healthcare). Relative binding of proteins to the lipids was quantitated by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD; <ext-link ext-link-type="uri" xlink:href="http://rsb.info.nih.gov/ij">http://rsb.info.nih.gov/ij</ext-link>). The data were normalized to the PtdIns(4,5)P<sub>2</sub> (for PIP Strips and Membrane Strips) or sulfatide (for SphingoStrips).</p></sec><sec id="s4-5"><title>Generation of liposomes and liposome pull-down assays</title><p>Liposomes were prepared as described previously (<xref ref-type="bibr" rid="bib76">Zhang et al., 2001</xref>) using lipids purchased from Avanti Polar Lipids (Alabaster, AL); L-α-phosphatidylcholine (PC, chicken egg), L-α-phosphatidyl-DL-glycerol (PG, chicken egg), L-α-phosphatidylethanolamine (PE, chicken egg), L-α-phosphatidylinositol (PI, bovine liver), L-α-phosphatidylserine (PS, porcine brain), and L-α-phosphatidylinositol-4,5 bisphosphate (PIP<sub>2</sub>, porcine brain). Lipids (dissolved in chloroform) and PIP<sub>2</sub> (dissolved in chloroform, methanol and water) were combined with the desired ratio of lipid components (PC:PE:PS:PI 50:30:10:10, PC:PE:PS:PI:PIP<sub>2</sub> 50:30:10:8:2, PC:PG 75:25, PC:PG:PIP<sub>2</sub> 75:20:5). The lipid mixture was dried under a stream of nitrogen gas followed by further drying under a vacuum for 3 hr. The lipid films were rehydrated in 500 µl of 50 mM HEPES (pH 7.0) to a concentration of 14 mg/ml for 2 hr with occasional vortexing. Lipid mixtures were freeze-thawed five times before sonicating for 8 min until the mixture cleared. Liposomes were washed twice in 50 mM HEPES (pH 7.0) prior to liposome binding assay. 50 µl of 14 mg/ml liposomes were incubated with 0.5 µg of NaD1 for 30 min at 25°C. Sample was pelleted by centrifugation at 16000×<italic>g</italic> and 30 µl of supernatant collected. The pellet was washed twice with 100 µl of 50 mM HEPES pH 7.0. Supernatant and pellet samples were analyzed for the presence of protein by SDS-PAGE and immunoblotting using a rabbit anti-NaD1 antibody as described for the protein-lipid overlay assay.</p></sec><sec id="s4-6"><title>Crystallographic methods</title><p>The NaD1:PIP<sub>2</sub> complexes were generated by mixing NaD1 at 10 mg/ml and PIP<sub>2</sub> at a molar ratio of 1:1.2. Crystals were grown in sitting drops at 20°C in 0.2 M ammonium sulfate, 7% PEG 3350, 32% MPD, and 0.1 M imidazole pH 7. Diffraction data were collected from crystals flash cooled in mother liquor at 100 K at the Australian Synchrotron (beamline MX2) and processed with Xds (<xref ref-type="bibr" rid="bib27">Kabsch, 2010</xref>). The structure was solved by molecular replacement with PHASER (<xref ref-type="bibr" rid="bib59">Storoni et al., 2004</xref>) using the structure of NaD1 (<xref ref-type="bibr" rid="bib37">Lay et al., 2012</xref>) as a search model. The final model was built with Coot (<xref ref-type="bibr" rid="bib14">Emsley and Cowtan, 2004</xref>) and refined with Phenix (<xref ref-type="bibr" rid="bib1">Adams et al., 2010</xref>) to a resolution of 1.6 Å. All data collection and refinement statistics are summarized in <xref ref-type="table" rid="tbl1">Table 1</xref>. Refinement yielded R<sub>work</sub> and R<sub>free</sub> values of 15.5% and 18.4%, respectively. All programs were accessed via the SBGrid suite (<xref ref-type="bibr" rid="bib44">Morin et al., 2013</xref>). The coordinates have been deposited in the Protein Data Bank (accession code 4CQK). Figures were prepared using PyMol.</p></sec><sec id="s4-7"><title>Cross-linking studies</title><p>NaD1 at 1 mg/ml (5 μl) was incubated with 2.3, 0.46, and 0.092 mM PIP<sub>2</sub> (5 μl) at room temperature for 30 min. Protein complexes were cross-linked through primary amino groups by the addition of 12.5 mM bis[sulfosuccinimidyl] suberate (BS<sup>3</sup>; 10 μl) in a buffer containing 20 mM sodium phosphate and 150 mM NaCl, pH 7.1, at room temperature for 30 min. Samples were reduced and denatured, and subjected to SDS-PAGE prior to Coomassie Brilliant Blue staining.</p></sec><sec id="s4-8"><title>Transmission electron microscopy (TEM)</title><p>TEM imaging was performed according to the procedure described by <xref ref-type="bibr" rid="bib1a">Adda et al. (2009)</xref>. In brief, samples (10 μl) were applied to 400-mesh copper grids coated with a thin layer of carbon for 2 min. Excess material was removed by blotting and samples were negatively stained twice with 10 μl of a 2% (wt/vol) uranyl acetate solution (Electron Microscopy Services, Hatfield, PA). The grids were air-dried and viewed using a JEOL JEM-2010 transmission electron microscope operated at 80 kV.</p></sec><sec id="s4-9"><title>Expression of rNaD1, rNaD1(R40E), and rNaD1(I37F) in <italic>Pichia pastoris</italic></title><p>Recombinant NaD1 and the point mutants rNaD1(R40E) and rNaD1(I37F) were cloned, expressed, and purified from the methylotropic yeast <italic>Pichia pastoris</italic> as described in <xref ref-type="bibr" rid="bib37">Lay et al. (2012)</xref>.</p></sec><sec id="s4-10"><title>Fungal growth inhibition assays</title><p>The ability of rNaD1, rNaD1(I37F), and rNaD1(R40E) to inhibit the growth of <italic>F. oxysporum</italic> f. sp. vasinfectum was assessed as described in <xref ref-type="bibr" rid="bib68">van der Weerden et al. (2008)</xref>, except that 6400 spores/well were used and growth was assessed after 48 hr. Each test was performed in triplicate. For data analysis, Prism 5 software (GraphPad Software Inc., San Diego, CA) was used to plot 4-parameter sigmoidal curves through the data.</p></sec><sec id="s4-11"><title>Cell lines</title><p>Human epithelial cervical cancer (HeLa) cells, prostate cancer (PC3) cells, and monocytic lymphoma (U937) cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA). All culture media were supplemented with 5–10% fetal calf serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin (Invitrogen). Cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO<sub>2</sub>. Adherent cell lines were detached from the flask by adding a mixture containing 0.25% trypsin and 0.5 μM EDTA (Invitrogen).</p></sec><sec id="s4-12"><title>Propidium iodide (PI) uptake assay</title><p>Flow cytometry-based PI uptake assay was performed to analyze the ability of NaD1 and related defensins to permeabilize tumor cells. Unless stated otherwise, U937 cells were suspended to 1 × 10<sup>6</sup> cells/ml in 0.1% BSA/RPMI-1640 and incubated with protein samples at 37°C for 30 min. Samples were added to PBS containing a final concentration of 1 μg/ml PI (Sigma-Aldrich, St Louis, MO) and placed on ice. Samples were then analyzed immediately using the BD FACSCanto II Flow Cytometer and BD FACSDiva software v6.1.1 (BD Biosciences, St Jose, CA). The resultant flow cytometry data were analyzed using FlowJo software v8.8.6 (Tree Star, Ashland, OR). Cells were gated appropriately based on forward scatter and side scatter and cell permeabilization was defined by PI-positive staining.</p></sec><sec id="s4-13"><title>FITC-dextran uptake assay</title><p>U937 cells and protein samples were prepared as per the PI uptake assay, with the exception that 100 μg/ml of FITC-dextran (4, 10, 20, or 40 kDa, Sigma-Aldrich) was present during the incubation at 37°C for 30 min. Samples were washed twice with 0.1% BSA/PBS to remove unbound FITC-dextran and added to PBS containing a final concentration of 1 μg/ml 7-aminoactinomycin D (7AAD) prior to analysis using the BD FACSCanto II Flow Cytometer and BD FACSDiva software. The resultant flow cytometry data were analyzed using FlowJo software.</p></sec><sec id="s4-14"><title>ATP bioluminescence assay</title><p>ATP bioluminescence assay (Roche, Mannheim, Germany) was used according to manufacturer’s instructions to measure the release of ATP from permeabilized tumor cells following treatment with defensins. Briefly, U937 cells were suspended to 1 × 10<sup>6</sup> cells/ml in 0.1% BSA/PBS and mixed with luciferase reagent at a ratio of 4:5 (vol:vol). The mixture of cells and luciferase reagent were added simultaneously to each well containing protein samples and luciferase activity was measured immediately on a SpectraMax M5e plate reader (Molecular Devices, Sunnyville, CA) at RT for 30 min with readings taken at 30 s intervals. The resultant data were analyzed using SoftMaxPro 5.2 software (Molecular Devices).</p></sec><sec id="s4-15"><title>Lactate dehydrogenase (LDH) release assay</title><p>LDH cytotoxicity assay kit II (Abcam, Cambridge, United Kingdom) was used according to manufacturer’s instructions to detect the release of the cytosolic enzyme, LDH, from U937 cells following treatment with defensins. Briefly, U937 cells were suspended at a cell concentration of 1 × 10<sup>6</sup> cells/ml in 0.1% BSA/RPMI-1640 and incubated with protein samples at 37°C for 30 min. Cells were then pelleted by centrifugation at 600×<italic>g</italic> and the supernatant was added to LDH reaction mix for 30 min at RT. The absorbance of the enzymatic product at 450 nm was measured using a SpectraMax M5e plate reader, with the resultant data analyzed using SoftMaxPro 5.2 software.</p></sec><sec id="s4-16"><title>MTT cell viability assay</title><p>Mammalian cells were seeded in quadruplicate into wells of a flat-bottomed 96-well microtitre plate (50 μl) at various densities starting at 2 × 10<sup>6</sup> cells/ml. Four wells containing complete culture medium alone were included in each assay as a background control. The microtitre plate was incubated overnight at 37°C under a humidified atmosphere containing 5% CO<sub>2</sub>/95% air, prior to the addition of complete culture medium (100 μl) to each well and further incubated at 37°C for 48 hr. Optimum cell densities (30–50% confluency) for cell viability assays were determined for each cell line by light microscopy. Mammalian cells were seeded in a 96-well microtitre plate (50 μl/well) at an optimum density determined in the cell optimization assay as above. Background control wells (n = 8) containing the same volume of complete culture medium were included in the assay. The microtitre plate was incubated overnight at 37°C, prior to the addition of NaD1 at various concentrations and the plate was incubated for a further 48 hr. The cell viability 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich) assay was performed as follows: the MTT solution (1 mg/ml) was added to each well (100 μl) and the plate incubated for 2–3 hr at 37°C under a humidified atmosphere containing 5% CO<sub>2</sub>/95% air. Subsequently, the media was removed and replaced with dimethyl sulfoxide (100 μl, DMSO, Sigma-Aldrich) and placed on a shaker for 5 min to dissolve the tetrazolium salts. Absorbance of each well was measured at 570 nm and the IC<sub>50</sub> values (the protein concentration to inhibit 50% of cell growth) were determined using OriginPro software v8.1.13.88 (OriginLab Corporation, Northampton, MA).</p></sec><sec id="s4-17"><title>Confocal laser scanning microscopy (CLSM)</title><p>Live imaging was performed on a Zeiss LSM 510 or LSM 780 confocal microscope using a 40× or 63× oil immersion objective in a 37°C/5% CO<sub>2</sub> atmosphere. Adherent cells were cultured on coverslips prior to imaging, while non-adherent cells were immobilized onto 10% poly-<sc>L</sc>-lysine-coated coverslips. All cell types were prepared for imaging in RPMI medium containing 0.1% BSA and 1–2 μg/ml PI. NaD1, BODIPY-NaD1, and FITC-Dextran (100 μg/ml) was added directly to the imaging chamber via a capillary tube. In certain experiments, cells were either stained with PKH67 (Sigma-Aldrich) or transfected with a plasmid construct for free GFP or GFP-PH(PLCδ) using Lipofectamine 2000 Reagent (Invitrogen) as per manufacturer’s instructions prior to imaging. The images were analyzed using ImageJ software or Zen software (Zeiss, Oberkochen, Germany). For quantification of CLSM experiments involving transfected cells, the effects of NaD1 on HeLa cells were observed over a 15 min timeframe from the time of NaD1 addition. Non-expressing cells were excluded from analysis.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank members of the Hulett and Kvansakul laboratories for their suggestions. We also thank S Cole for assistance with the purification of native NaD1; A Bojceski for assistance with the protein-lipid overlay assays; M Wong for assistance with flow cytometry; J Dyson and C Mitchell for suggestions and providing the construct for GFP-PH(PLCδ); T Brown for assistance with CLSM; the Bio21 Collaborative Crystallisation Centre for assistance with crystallization; MX2 staff at the Australian Synchrotron for assistance with X-ray diffraction data collection; SBGrid consortium for software support. This work was supported by Balmoral Australia Pty Ltd and Hexima Ltd.</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>NLW: Head of Technology Commercialisation Hexima Ltd.</p></fn><fn fn-type="conflict" id="conf2"><p>MAA: Chief Science Officer Hexima Ltd.</p></fn><fn fn-type="conflict" id="conf3"><p>MDH: Vice President of Research Hexima Ltd.</p></fn><fn fn-type="conflict" id="conf4"><p>The other 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>IKHP, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>AAB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>FTL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>MK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>MDH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con6"><p>GDM, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con7"><p>CGA, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con8"><p>JAEP, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con9"><p>TKP, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con10"><p>GFR, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con11"><p>JAW, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con12"><p>PKV, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con13"><p>NLW, 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="con14"><p>MAA, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</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 dataset was 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>Lay</surname><given-names>FT</given-names></name>, <name><surname>Mills</surname><given-names>GM</given-names></name>, <name><surname>Poon</surname><given-names>IKH</given-names></name>, <name><surname>Baxter</surname><given-names>AA</given-names></name>, <name><surname>Hulett</surname><given-names>MD</given-names></name>, <name><surname>Kvansakul</surname><given-names>M</given-names></name>, <year>2014</year><x>, </x><source>Crystal structure of ligand-bound NaD1</source><x>, </x><object-id pub-id-type="art-access-id">4CQK</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4CQK">http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4CQK</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>Adams</surname><given-names>PD</given-names></name><name><surname>Afonine</surname><given-names>PV</given-names></name><name><surname>Bunkoczi</surname><given-names>G</given-names></name><name><surname>Chen</surname><given-names>VB</given-names></name><name><surname>Davis</surname><given-names>IW</given-names></name><name><surname>Echols</surname><given-names>N</given-names></name><name><surname>Headd</surname><given-names>JJ</given-names></name><name><surname>Hung</surname><given-names>LW</given-names></name><name><surname>Kapral</surname><given-names>GJ</given-names></name><name><surname>Grosse-Kunstleve</surname><given-names>RW</given-names></name><name><surname>McCoy</surname><given-names>AJ</given-names></name><name><surname>Moriarty</surname><given-names>NW</given-names></name><name><surname>Oeffner</surname><given-names>R</given-names></name><name><surname>Read</surname><given-names>RJ</given-names></name><name><surname>Richardson</surname><given-names>DC</given-names></name><name><surname>Richardson</surname><given-names>JS</given-names></name><name><surname>Terwilliger</surname><given-names>TC</given-names></name><name><surname>Zwart</surname><given-names>PH</given-names></name></person-group><year>2010</year><article-title>PHENIX: a comprehensive Python-based system for macromolecular structure solution</article-title><source>Acta Crystallographica Section D, Biological Crystallography</source><volume>66</volume><fpage>213</fpage><lpage>221</lpage><pub-id pub-id-type="doi">10.1107/S0907444909052925</pub-id></element-citation></ref><ref id="bib1a"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Adda</surname><given-names>CG</given-names></name><name><surname>Murphy</surname><given-names>VJ</given-names></name><name><surname>Sunde</surname><given-names>M</given-names></name><name><surname>Waddington</surname><given-names>LJ</given-names></name><name><surname>Schloegel</surname><given-names>J</given-names></name><name><surname>Talbo</surname><given-names>GH</given-names></name><name><surname>Vingas</surname><given-names>K</given-names></name><name><surname>Kienzle</surname><given-names>V</given-names></name><name><surname>Masciantonio</surname><given-names>R</given-names></name><name><surname>Howlett</surname><given-names>GJ</given-names></name><name><surname>Hodder</surname><given-names>AN</given-names></name><name><surname>Foley</surname><given-names>M</given-names></name><name><surname>Anders</surname><given-names>RF</given-names></name></person-group><year>2009</year><article-title><italic>Plasmodium falciparum</italic> merozoite surface protein 2 is unstructured and forms amyloid-like fibrils</article-title><source>Molecular and Biochemical Parasitology</source><volume>166</volume><fpage>159</fpage><lpage>71</lpage><pub-id pub-id-type="doi">10.1016/j.molbiopara.2009.03.012</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ashida</surname><given-names>H</given-names></name><name><surname>Ogawa</surname><given-names>M</given-names></name><name><surname>Kim</surname><given-names>M</given-names></name><name><surname>Mimuro</surname><given-names>H</given-names></name><name><surname>Sasakawa</surname><given-names>C</given-names></name></person-group><year>2011</year><article-title>Bacteria and host interactions in the gut epithelial barrier</article-title><source>Nature Chemical Biology</source><volume>8</volume><fpage>36</fpage><lpage>45</lpage><pub-id pub-id-type="doi">10.1038/nchembio.741</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bravo</surname><given-names>J</given-names></name><name><surname>Karathanassis</surname><given-names>D</given-names></name><name><surname>Pacold</surname><given-names>CM</given-names></name><name><surname>Pacold</surname><given-names>ME</given-names></name><name><surname>Ellson</surname><given-names>CD</given-names></name><name><surname>Anderson</surname><given-names>KE</given-names></name><name><surname>Butler</surname><given-names>PJ</given-names></name><name><surname>Lavenir</surname><given-names>I</given-names></name><name><surname>Perisic</surname><given-names>O</given-names></name><name><surname>Hawkins</surname><given-names>PT</given-names></name><name><surname>Stephens</surname><given-names>L</given-names></name><name><surname>Williams</surname><given-names>RL</given-names></name></person-group><year>2001</year><article-title>The crystal structure of the PX domain from p40(phox) bound to phosphatidylinositol 3-phosphate</article-title><source>Molecular Cell</source><volume>8</volume><fpage>829</fpage><lpage>839</lpage><pub-id pub-id-type="doi">10.1016/S1097-2765(01)00372-0</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Broberg</surname><given-names>CA</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Gonzalez</surname><given-names>H</given-names></name><name><surname>Laskowski-Arce</surname><given-names>MA</given-names></name><name><surname>Orth</surname><given-names>K</given-names></name></person-group><year>2010</year><article-title>A Vibrio effector protein is an inositol phosphatase and disrupts host cell membrane integrity</article-title><source>Science</source><volume>329</volume><fpage>1660</fpage><lpage>1662</lpage><pub-id pub-id-type="doi">10.1126/science.1192850</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brogden</surname><given-names>KA</given-names></name></person-group><year>2005</year><article-title>Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?</article-title><source>Nature Review Microbiology</source><volume>3</volume><fpage>238</fpage><lpage>250</lpage><pub-id pub-id-type="doi">10.1038/nrmicro1098</pub-id></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Carvalho</surname><given-names>ADO</given-names></name><name><surname>Gomes</surname><given-names>VM</given-names></name></person-group><year>2009</year><article-title>Plant defensins—Prospects for the biological functions and biotechnological properties</article-title><source>Peptides</source><volume>30</volume><fpage>1007</fpage><lpage>1020</lpage><pub-id pub-id-type="doi">10.1016/j.peptides.2009.01.018</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chaudhary</surname><given-names>J</given-names></name><name><surname>Munshi</surname><given-names>M</given-names></name></person-group><year>1995</year><article-title>Scanning electron microscopic analysis of breast aspirates</article-title><source>Cytopathology</source><volume>6</volume><fpage>162</fpage><lpage>167</lpage><pub-id pub-id-type="doi">10.1111/j.1365-2303.1995.tb00469.x</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chu</surname><given-names>H</given-names></name><name><surname>Pazgier</surname><given-names>M</given-names></name><name><surname>Jung</surname><given-names>G</given-names></name><name><surname>Nuccio</surname><given-names>SP</given-names></name><name><surname>Castillo</surname><given-names>PA</given-names></name><name><surname>De Jong</surname><given-names>MF</given-names></name><name><surname>Winter</surname><given-names>MG</given-names></name><name><surname>Winter</surname><given-names>SE</given-names></name><name><surname>Wehkamp</surname><given-names>J</given-names></name><name><surname>Shen</surname><given-names>B</given-names></name><name><surname>Salzman</surname><given-names>NH</given-names></name><name><surname>Underwood</surname><given-names>MA</given-names></name><name><surname>Tsolis</surname><given-names>RM</given-names></name><name><surname>Young</surname><given-names>GM</given-names></name><name><surname>Lu</surname><given-names>W</given-names></name><name><surname>Lehrer</surname><given-names>RI</given-names></name><name><surname>Bäumler</surname><given-names>AJ</given-names></name><name><surname>Bevins</surname><given-names>CL</given-names></name></person-group><year>2012</year><article-title>Human alpha-defensin 6 promotes mucosal innate immunity through self-assembled peptide nanonets</article-title><source>Science</source><volume>337</volume><fpage>477</fpage><lpage>481</lpage><pub-id pub-id-type="doi">10.1126/science.1218831</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cruciani</surname><given-names>RA</given-names></name><name><surname>Barker</surname><given-names>JL</given-names></name><name><surname>Zasloff</surname><given-names>M</given-names></name><name><surname>Chen</surname><given-names>HC</given-names></name><name><surname>Colamonici</surname><given-names>O</given-names></name></person-group><year>1991</year><article-title>Antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation</article-title><source>Proceedings of the National Academy of Sciences of the United States of America</source><volume>88</volume><fpage>3792</fpage><lpage>3796</lpage><pub-id pub-id-type="doi">10.1073/pnas.88.9.3792</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>De Coninck</surname><given-names>B</given-names></name><name><surname>Cammue</surname><given-names>BPA</given-names></name><name><surname>Thevissen</surname><given-names>K</given-names></name></person-group><year>2013</year><article-title>Modes of antifungal action and in planta functions of plant defensins and defensin-like peptides</article-title><source>Fungal Biology Reviews</source><volume>26</volume><fpage>109</fpage><lpage>120</lpage><pub-id pub-id-type="doi">10.1016/j.fbr.2012.10.002</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>de Paula</surname><given-names>VS</given-names></name><name><surname>Razzera</surname><given-names>G</given-names></name><name><surname>Barreto-Bergter</surname><given-names>E</given-names></name><name><surname>Almeida</surname><given-names>FC</given-names></name><name><surname>Valente</surname><given-names>AP</given-names></name></person-group><year>2011</year><article-title>Portrayal of complex dynamic properties of sugarcane defensin 5 by NMR: multiple motions associated with membrane interaction</article-title><source>Structure</source><volume>19</volume><fpage>26</fpage><lpage>36</lpage><pub-id pub-id-type="doi">10.1016/j.str.2010.11.011</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>De Samblanx</surname><given-names>GW</given-names></name><name><surname>Goderis</surname><given-names>IJ</given-names></name><name><surname>Thevissen</surname><given-names>K</given-names></name><name><surname>Raemaekers</surname><given-names>R</given-names></name><name><surname>Fant</surname><given-names>F</given-names></name><name><surname>Borremans</surname><given-names>F</given-names></name><name><surname>Acland</surname><given-names>DP</given-names></name><name><surname>Osborn</surname><given-names>RW</given-names></name><name><surname>Patel</surname><given-names>S</given-names></name><name><surname>Broekaert</surname><given-names>WF</given-names></name></person-group><year>1997</year><article-title>Mutational analysis of a plant defensin from radish (Raphanus sativus L.) reveals two adjacent sites important for antifungal activity</article-title><source>The Journal of Biological Chemistry</source><volume>272</volume><fpage>1171</fpage><lpage>1179</lpage><pub-id pub-id-type="doi">10.1074/jbc.272.2.1171</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Di Paolo</surname><given-names>G</given-names></name><name><surname>De Camilli</surname><given-names>P</given-names></name></person-group><year>2006</year><article-title>Phosphoinositides in cell regulation and membrane dynamics</article-title><source>Nature</source><volume>443</volume><fpage>651</fpage><lpage>657</lpage><pub-id pub-id-type="doi">10.1038/nature05185</pub-id></element-citation></ref><ref id="bib14"><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 Crystallographica Section D, Biological Crystallography</source><volume>60</volume><fpage>2126</fpage><lpage>2132</lpage><pub-id pub-id-type="doi">10.1107/S0907444904019158</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Garcia-Saez</surname><given-names>AJ</given-names></name><name><surname>Buschhorn</surname><given-names>SB</given-names></name><name><surname>Keller</surname><given-names>H</given-names></name><name><surname>Anderluh</surname><given-names>G</given-names></name><name><surname>Simons</surname><given-names>K</given-names></name><name><surname>Schwille</surname><given-names>P</given-names></name></person-group><year>2011</year><article-title>Oligomerization and pore formation by equinatoxin II inhibit endocytosis and lead to plasma membrane reorganization</article-title><source>The Journal of Biological Chemistry</source><volume>286</volume><fpage>37768</fpage><lpage>37777</lpage><pub-id pub-id-type="doi">10.1074/jbc.M111.281592</pub-id></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Goncalves</surname><given-names>S</given-names></name><name><surname>Teixeira</surname><given-names>A</given-names></name><name><surname>Abade</surname><given-names>J</given-names></name><name><surname>De Medeiros</surname><given-names>LN</given-names></name><name><surname>Kurtenbach</surname><given-names>E</given-names></name><name><surname>Santos</surname><given-names>NC</given-names></name></person-group><year>2012</year><article-title>Evaluation of the membrane lipid selectivity of the pea defensin Psd1</article-title><source>Biochimica et Biophysica Acta</source><volume>1818</volume><fpage>1420</fpage><lpage>1426</lpage><pub-id pub-id-type="doi">10.1016/j.bbamem.2012.02.012</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hadders</surname><given-names>MA</given-names></name><name><surname>Beringer</surname><given-names>DX</given-names></name><name><surname>Gros</surname><given-names>P</given-names></name></person-group><year>2007</year><article-title>Structure of C8alpha-MACPF reveals mechanism of membrane attack in complement immune defense</article-title><source>Science</source><volume>317</volume><fpage>1552</fpage><lpage>1554</lpage><pub-id pub-id-type="doi">10.1126/science.1147103</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hancock</surname><given-names>RE</given-names></name><name><surname>Lehrer</surname><given-names>R</given-names></name></person-group><year>1998</year><article-title>Cationic peptides: a new source of antibiotics</article-title><source>Trends in Biotechnology</source><volume>16</volume><fpage>82</fpage><lpage>88</lpage><pub-id pub-id-type="doi">10.1016/S0167-7799(97)01156-6</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hancock</surname><given-names>RE</given-names></name><name><surname>Sahl</surname><given-names>HG</given-names></name></person-group><year>2006</year><article-title>Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies</article-title><source>Nature Biotechnology</source><volume>24</volume><fpage>1551</fpage><lpage>1557</lpage><pub-id pub-id-type="doi">10.1038/nbt1267</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hayes</surname><given-names>BM</given-names></name><name><surname>Bleackley</surname><given-names>MR</given-names></name><name><surname>Wiltshire</surname><given-names>JL</given-names></name><name><surname>Anderson</surname><given-names>MA</given-names></name><name><surname>Traven</surname><given-names>A</given-names></name><name><surname>Van Der Weerden</surname><given-names>NL</given-names></name></person-group><year>2013</year><article-title>Identification and mechanism of action of the plant defensin NaD1 as a new member of the antifungal drug arsenal against <italic>Candida albicans</italic></article-title><source>Antimicrobial Agents and Chemotherapy</source><volume>57</volume><fpage>3667</fpage><lpage>3675</lpage><pub-id pub-id-type="doi">10.1128/AAC.00365-13</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Henriques</surname><given-names>ST</given-names></name><name><surname>Melo</surname><given-names>MN</given-names></name><name><surname>Castanho</surname><given-names>MA</given-names></name></person-group><year>2006</year><article-title>Cell-penetrating peptides and antimicrobial peptides: how different are they?</article-title><source>Biochemical Journal</source><volume>399</volume><fpage>1</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1042/BJ20061100</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hill</surname><given-names>CP</given-names></name><name><surname>Yee</surname><given-names>J</given-names></name><name><surname>Selsted</surname><given-names>ME</given-names></name><name><surname>Eisenberg</surname><given-names>D</given-names></name></person-group><year>1991</year><article-title>Crystal structure of defensin HNP-3, an amphiphilic dimer: mechanisms of membrane permeabilization</article-title><source>Science</source><volume>251</volume><fpage>1481</fpage><lpage>1485</lpage><pub-id pub-id-type="doi">10.1126/science.2006422</pub-id></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hoover</surname><given-names>DM</given-names></name><name><surname>Rajashankar</surname><given-names>KR</given-names></name><name><surname>Blumenthal</surname><given-names>R</given-names></name><name><surname>Puri</surname><given-names>A</given-names></name><name><surname>Oppenheim</surname><given-names>JJ</given-names></name><name><surname>Chertov</surname><given-names>O</given-names></name><name><surname>Lubkowski</surname><given-names>J</given-names></name></person-group><year>2000</year><article-title>The structure of human beta-defensin-2 shows evidence of higher order oligomerization</article-title><source>The Journal of Biological Chemistry</source><volume>275</volume><fpage>32911</fpage><lpage>32918</lpage><pub-id pub-id-type="doi">10.1074/jbc.M006098200</pub-id></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hsu</surname><given-names>ST</given-names></name><name><surname>Breukink</surname><given-names>E</given-names></name><name><surname>Tischenko</surname><given-names>E</given-names></name><name><surname>Lutters</surname><given-names>MA</given-names></name><name><surname>De Kruijff</surname><given-names>B</given-names></name><name><surname>Kaptein</surname><given-names>R</given-names></name><name><surname>Bonvin</surname><given-names>AM</given-names></name><name><surname>van Nuland</surname><given-names>NA</given-names></name></person-group><year>2004</year><article-title>The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics</article-title><source>Nature Structure Molecular Biology</source><volume>11</volume><fpage>963</fpage><lpage>967</lpage><pub-id pub-id-type="doi">10.1038/nsmb830</pub-id></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ino</surname><given-names>Y</given-names></name><name><surname>Gotoh</surname><given-names>M</given-names></name><name><surname>Sakamoto</surname><given-names>M</given-names></name><name><surname>Tsukagoshi</surname><given-names>K</given-names></name><name><surname>Hirohashi</surname><given-names>S</given-names></name></person-group><year>2002</year><article-title>Dysadherin, a cancer-associated cell membrane glycoprotein, down-regulates E-cadherin and promotes metastasis</article-title><source>Proceedings of the National Academy of Sciences of the United States of America</source><volume>99</volume><fpage>365</fpage><lpage>370</lpage><pub-id pub-id-type="doi">10.1073/pnas.012425299</pub-id></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Janssen</surname><given-names>BJ</given-names></name><name><surname>Schirra</surname><given-names>HJ</given-names></name><name><surname>Lay</surname><given-names>FT</given-names></name><name><surname>Anderson</surname><given-names>MA</given-names></name><name><surname>Craik</surname><given-names>DJ</given-names></name></person-group><year>2003</year><article-title>Structure of <italic>Petunia hybrida</italic> defensin 1, a novel plant defensin with five disulfide bonds</article-title><source>Biochemistry</source><volume>42</volume><fpage>8214</fpage><lpage>8222</lpage><pub-id pub-id-type="doi">10.1021/bi034379o</pub-id></element-citation></ref><ref id="bib27"><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 Crystallographica Section D, Biological Crystallography</source><volume>66</volume><fpage>125</fpage><lpage>132</lpage><pub-id pub-id-type="doi">10.1107/S0907444909047337</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kale</surname><given-names>SD</given-names></name><name><surname>Gu</surname><given-names>B</given-names></name><name><surname>Capelluto</surname><given-names>DG</given-names></name><name><surname>Dou</surname><given-names>D</given-names></name><name><surname>Feldman</surname><given-names>E</given-names></name><name><surname>Rumore</surname><given-names>A</given-names></name><name><surname>Arredondo</surname><given-names>FD</given-names></name><name><surname>Hanlon</surname><given-names>R</given-names></name><name><surname>Fudal</surname><given-names>I</given-names></name><name><surname>Rouxel</surname><given-names>T</given-names></name><name><surname>Lawrence</surname><given-names>CB</given-names></name><name><surname>Shan</surname><given-names>W</given-names></name><name><surname>Tyler</surname><given-names>BM</given-names></name></person-group><year>2010</year><article-title>External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells</article-title><source>Cell</source><volume>142</volume><fpage>284</fpage><lpage>295</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2010.06.008</pub-id></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Keller</surname><given-names>H</given-names></name><name><surname>Lorizate</surname><given-names>M</given-names></name><name><surname>Schwille</surname><given-names>P</given-names></name></person-group><year>2009</year><article-title>PI(4,5)P2 degradation promotes the formation of cytoskeleton-free model membrane systems</article-title><source>Chemphyschem</source><volume>10</volume><fpage>2805</fpage><lpage>2812</lpage><pub-id pub-id-type="doi">10.1002/cphc.200900598</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kufe</surname><given-names>DW</given-names></name></person-group><year>2009</year><article-title>Mucins in cancer: function, prognosis and therapy</article-title><source>Nature Review Cancer</source><volume>9</volume><fpage>874</fpage><lpage>885</lpage><pub-id pub-id-type="doi">10.1038/nrc2761</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kutateladze</surname><given-names>T</given-names></name><name><surname>Overduin</surname><given-names>M</given-names></name></person-group><year>2001</year><article-title>Structural mechanism of endosome docking by the FYVE domain</article-title><source>Science</source><volume>291</volume><fpage>1793</fpage><lpage>1796</lpage><pub-id pub-id-type="doi">10.1126/science.291.5509.1793</pub-id></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lai</surname><given-names>Y</given-names></name><name><surname>Gallo</surname><given-names>RL</given-names></name></person-group><year>2009</year><article-title>AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense</article-title><source>Trends in Immunology</source><volume>30</volume><fpage>131</fpage><lpage>141</lpage><pub-id pub-id-type="doi">10.1016/j.it.2008.12.003</pub-id></element-citation></ref><ref id="bib33"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Law</surname><given-names>RH</given-names></name><name><surname>Lukoyanova</surname><given-names>N</given-names></name><name><surname>Voskoboinik</surname><given-names>I</given-names></name><name><surname>Caradoc-Davies</surname><given-names>TT</given-names></name><name><surname>Baran</surname><given-names>K</given-names></name><name><surname>Dunstone</surname><given-names>MA</given-names></name><name><surname>D’Angelo</surname><given-names>ME</given-names></name><name><surname>Orlova</surname><given-names>EV</given-names></name><name><surname>Coulibaly</surname><given-names>F</given-names></name><name><surname>Verschoor</surname><given-names>S</given-names></name><name><surname>Browne</surname><given-names>KA</given-names></name><name><surname>Ciccone</surname><given-names>A</given-names></name><name><surname>Kuiper</surname><given-names>MJ</given-names></name><name><surname>Bird</surname><given-names>PI</given-names></name><name><surname>Trapani</surname><given-names>JA</given-names></name><name><surname>Saibil</surname><given-names>HR</given-names></name><name><surname>Whisstock</surname><given-names>JC</given-names></name></person-group><year>2010</year><article-title>The structural basis for membrane binding and pore formation by lymphocyte perforin</article-title><source>Nature</source><volume>468</volume><fpage>447</fpage><lpage>451</lpage><pub-id pub-id-type="doi">10.1038/nature09518</pub-id></element-citation></ref><ref id="bib34"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lay</surname><given-names>FT</given-names></name><name><surname>Brugliera</surname><given-names>F</given-names></name><name><surname>Anderson</surname><given-names>MA</given-names></name></person-group><year>2003a</year><article-title>Isolation and properties of floral defensins from ornamental tobacco and petunia</article-title><source>Plant Physiology</source><volume>131</volume><fpage>1283</fpage><lpage>1293</lpage><pub-id pub-id-type="doi">10.1104/pp.102.016626</pub-id></element-citation></ref><ref id="bib35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lay</surname><given-names>FT</given-names></name><name><surname>Schirra</surname><given-names>HJ</given-names></name><name><surname>Scanlon</surname><given-names>MJ</given-names></name><name><surname>Anderson</surname><given-names>MA</given-names></name><name><surname>Craik</surname><given-names>DJ</given-names></name></person-group><year>2003b</year><article-title>The three-dimensional solution structure of NaD1, a new floral defensin from <italic>Nicotiana alata</italic> and its application to a homology model of the crop defense protein alfAFP</article-title><source>Journal of Molecular Biology</source><volume>325</volume><fpage>175</fpage><lpage>188</lpage><pub-id pub-id-type="doi">10.1016/S0022-2836(02)01103-8</pub-id></element-citation></ref><ref id="bib36"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lay</surname><given-names>FT</given-names></name><name><surname>Anderson</surname><given-names>MA</given-names></name></person-group><year>2005</year><article-title>Defensins - components of the innate immune system in plants</article-title><source>Current Protein & Peptide Science</source><volume>6</volume><fpage>85</fpage><lpage>101</lpage><pub-id pub-id-type="doi">10.2174/1389203053027575</pub-id></element-citation></ref><ref id="bib37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lay</surname><given-names>FT</given-names></name><name><surname>Mills</surname><given-names>GD</given-names></name><name><surname>Poon</surname><given-names>IK</given-names></name><name><surname>Cowieson</surname><given-names>NP</given-names></name><name><surname>Kirby</surname><given-names>N</given-names></name><name><surname>Baxter</surname><given-names>AA</given-names></name><name><surname>van der Weerden</surname><given-names>NL</given-names></name><name><surname>Dogovski</surname><given-names>C</given-names></name><name><surname>Perugini</surname><given-names>MA</given-names></name><name><surname>Anderson</surname><given-names>MA</given-names></name><name><surname>Kvansakul</surname><given-names>M</given-names></name><name><surname>Hulett</surname><given-names>MD</given-names></name></person-group><year>2012</year><article-title>Dimerization of plant defensin NaD1 enhances its antifungal activity</article-title><source>The Journal of Biological Chemistry</source><volume>287</volume><fpage>19961</fpage><lpage>19972</lpage><pub-id pub-id-type="doi">10.1074/jbc.M111.331009</pub-id></element-citation></ref><ref id="bib38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lemmon</surname><given-names>MA</given-names></name></person-group><year>2008</year><article-title>Membrane recognition by phospholipid-binding domains</article-title><source>Nature Reviews Molecular Cell Biology</source><volume>9</volume><fpage>99</fpage><lpage>111</lpage><pub-id pub-id-type="doi">10.1038/nrm2328</pub-id></element-citation></ref><ref id="bib39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lichtenstein</surname><given-names>A</given-names></name><name><surname>Ganz</surname><given-names>T</given-names></name><name><surname>Selsted</surname><given-names>ME</given-names></name><name><surname>Lehrer</surname><given-names>RI</given-names></name></person-group><year>1986</year><article-title>In vitro tumor cell cytolysis mediated by peptide defensins of human and rabbit granulocytes</article-title><source>Blood</source><volume>68</volume><fpage>1407</fpage><lpage>1410</lpage></element-citation></ref><ref id="bib40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lietzke</surname><given-names>SE</given-names></name><name><surname>Bose</surname><given-names>S</given-names></name><name><surname>Cronin</surname><given-names>T</given-names></name><name><surname>Klarlund</surname><given-names>J</given-names></name><name><surname>Chawla</surname><given-names>A</given-names></name><name><surname>Czech</surname><given-names>MP</given-names></name><name><surname>Lambright</surname><given-names>DG</given-names></name></person-group><year>2000</year><article-title>Structural basis of 3-phosphoinositide recognition by pleckstrin homology domains</article-title><source>Molecular Cell</source><volume>6</volume><fpage>385</fpage><lpage>394</lpage><pub-id pub-id-type="doi">10.1016/S1097-2765(00)00038-1</pub-id></element-citation></ref><ref id="bib41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lin</surname><given-names>KF</given-names></name><name><surname>Lee</surname><given-names>TR</given-names></name><name><surname>Tsai</surname><given-names>PH</given-names></name><name><surname>Hsu</surname><given-names>MP</given-names></name><name><surname>Chen</surname><given-names>CS</given-names></name><name><surname>Lyu</surname><given-names>PC</given-names></name></person-group><year>2007</year><article-title>Structure-based protein engineering for alpha-amylase inhibitory activity of plant defensin</article-title><source>Proteins</source><volume>68</volume><fpage>530</fpage><lpage>540</lpage><pub-id pub-id-type="doi">10.1002/prot.21378</pub-id></element-citation></ref><ref id="bib42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mader</surname><given-names>JS</given-names></name><name><surname>Hoskin</surname><given-names>DW</given-names></name></person-group><year>2006</year><article-title>Cationic antimicrobial peptides as novel cytotoxic agents for cancer treatment</article-title><source>Expert Opinion on Investigational Drugs</source><volume>15</volume><fpage>933</fpage><lpage>946</lpage><pub-id pub-id-type="doi">10.1517/13543784.15.8.933</pub-id></element-citation></ref><ref id="bib43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McLaughlin</surname><given-names>S</given-names></name><name><surname>Murray</surname><given-names>D</given-names></name></person-group><year>2005</year><article-title>Plasma membrane phosphoinositide organization by protein electrostatics</article-title><source>Nature</source><volume>438</volume><fpage>605</fpage><lpage>611</lpage><pub-id pub-id-type="doi">10.1038/nature04398</pub-id></element-citation></ref><ref id="bib44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Morin</surname><given-names>A</given-names></name><name><surname>Eisenbraun</surname><given-names>B</given-names></name><name><surname>Key</surname><given-names>J</given-names></name><name><surname>Sanschagrin</surname><given-names>PC</given-names></name><name><surname>Timony</surname><given-names>MA</given-names></name><name><surname>Ottaviano</surname><given-names>M</given-names></name><name><surname>Sliz</surname><given-names>P</given-names></name></person-group><year>2013</year><article-title>Collaboration gets the most out of software</article-title><source>eLife</source><volume>2</volume><fpage>e01456</fpage><pub-id pub-id-type="doi">10.7554/eLife.01456</pub-id></element-citation></ref><ref id="bib45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Niebuhr</surname><given-names>K</given-names></name><name><surname>Giuriato</surname><given-names>S</given-names></name><name><surname>Pedron</surname><given-names>T</given-names></name><name><surname>Philpott</surname><given-names>DJ</given-names></name><name><surname>Gaits</surname><given-names>F</given-names></name><name><surname>Sable</surname><given-names>J</given-names></name><name><surname>Sheetz</surname><given-names>MP</given-names></name><name><surname>Parsot</surname><given-names>C</given-names></name><name><surname>Sansonetti</surname><given-names>PJ</given-names></name><name><surname>Payrastre</surname><given-names>B</given-names></name></person-group><year>2002</year><article-title>Conversion of PtdIns(4,5)P(2) into PtdIns(5)P by the <italic>S.flexneri</italic> effector IpgD reorganizes host cell morphology</article-title><source>The EMBO Journal</source><volume>21</volume><fpage>5069</fpage><lpage>5078</lpage><pub-id pub-id-type="doi">10.1093/emboj/cdf522</pub-id></element-citation></ref><ref id="bib46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Patel</surname><given-names>KK</given-names></name><name><surname>Poon</surname><given-names>IK</given-names></name><name><surname>Talbo</surname><given-names>GH</given-names></name><name><surname>Perugini</surname><given-names>MA</given-names></name><name><surname>Taylor</surname><given-names>NL</given-names></name><name><surname>Ralph</surname><given-names>TJ</given-names></name><name><surname>Hoogenraad</surname><given-names>NJ</given-names></name><name><surname>Hulett</surname><given-names>MD</given-names></name></person-group><year>2013</year><article-title>New method for purifying histidine-rich glycoprotein from human plasma redefines its functional properties</article-title><source>IUBMB Life</source><volume>65</volume><fpage>550</fpage><lpage>563</lpage><pub-id pub-id-type="doi">10.1002/iub.1168</pub-id></element-citation></ref><ref id="bib47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Poon</surname><given-names>IK</given-names></name><name><surname>Hulett</surname><given-names>MD</given-names></name><name><surname>Parish</surname><given-names>CR</given-names></name></person-group><year>2010</year><article-title>Histidine-rich glycoprotein is a novel plasma pattern recognition molecule that recruits IgG to facilitate necrotic cell clearance via FcgammaRI on phagocytes</article-title><source>Blood</source><volume>115</volume><fpage>2473</fpage><lpage>2482</lpage><pub-id pub-id-type="doi">10.1182/blood-2009-07-234013</pub-id></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ramamoorthy</surname><given-names>V</given-names></name><name><surname>Cahoon</surname><given-names>EB</given-names></name><name><surname>Li</surname><given-names>J</given-names></name><name><surname>Thokala</surname><given-names>M</given-names></name><name><surname>Minto</surname><given-names>RE</given-names></name><name><surname>Shah</surname><given-names>DM</given-names></name></person-group><year>2007</year><article-title>Glucosylceramide synthase is essential for alfalfa defensin-mediated growth inhibition but not for pathogenicity of Fusarium graminearum</article-title><source>Molecular Microbiology</source><volume>66</volume><fpage>771</fpage><lpage>786</lpage><pub-id pub-id-type="doi">10.1111/j.1365-2958.2007.05955.x</pub-id></element-citation></ref><ref id="bib49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ran</surname><given-names>S</given-names></name><name><surname>Downes</surname><given-names>A</given-names></name><name><surname>Thorpe</surname><given-names>PE</given-names></name></person-group><year>2002</year><article-title>Increased exposure of anionic phospholipids on the surface of tumor blood vessels</article-title><source>Cancer Research</source><volume>62</volume><fpage>6132</fpage><lpage>6140</lpage></element-citation></ref><ref id="bib50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Raucher</surname><given-names>D</given-names></name><name><surname>Stauffer</surname><given-names>T</given-names></name><name><surname>Chen</surname><given-names>W</given-names></name><name><surname>Shen</surname><given-names>K</given-names></name><name><surname>Guo</surname><given-names>S</given-names></name><name><surname>York</surname><given-names>JD</given-names></name><name><surname>Sheetz</surname><given-names>MP</given-names></name><name><surname>Meyer</surname><given-names>T</given-names></name></person-group><year>2000</year><article-title>Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion</article-title><source>Cell</source><volume>100</volume><fpage>221</fpage><lpage>228</lpage><pub-id pub-id-type="doi">10.1016/S0092-8674(00)81560-3</pub-id></element-citation></ref><ref id="bib51"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rossjohn</surname><given-names>J</given-names></name><name><surname>Feil</surname><given-names>SC</given-names></name><name><surname>Mckinstry</surname><given-names>WJ</given-names></name><name><surname>Tweten</surname><given-names>RK</given-names></name><name><surname>Parker</surname><given-names>MW</given-names></name></person-group><year>1997</year><article-title>Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form</article-title><source>Cell</source><volume>89</volume><fpage>685</fpage><lpage>692</lpage><pub-id pub-id-type="doi">10.1016/S0092-8674(00)80251-2</pub-id></element-citation></ref><ref id="bib52"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sagaram</surname><given-names>US</given-names></name><name><surname>Pandurangi</surname><given-names>R</given-names></name><name><surname>Kaur</surname><given-names>J</given-names></name><name><surname>Smith</surname><given-names>TJ</given-names></name><name><surname>Shah</surname><given-names>DM</given-names></name></person-group><year>2011</year><article-title>Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum</article-title><source>PLOS ONE</source><volume>6</volume><fpage>e18550</fpage></element-citation></ref><ref id="bib53"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sahl</surname><given-names>HG</given-names></name><name><surname>Bierbaum</surname><given-names>G</given-names></name></person-group><year>1998</year><article-title>Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from Gram-positive bacteria</article-title><source>Annual Review of Microbiology</source><volume>52</volume><fpage>41</fpage><lpage>79</lpage><pub-id pub-id-type="doi">10.1146/annurev.micro.52.1.41</pub-id></element-citation></ref><ref id="bib54"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schneider</surname><given-names>T</given-names></name><name><surname>Kruse</surname><given-names>T</given-names></name><name><surname>Wimmer</surname><given-names>R</given-names></name><name><surname>Wiedemann</surname><given-names>I</given-names></name><name><surname>Sass</surname><given-names>V</given-names></name><name><surname>Pag</surname><given-names>U</given-names></name><name><surname>Jansen</surname><given-names>A</given-names></name><name><surname>Nielsen</surname><given-names>AK</given-names></name><name><surname>Mygind</surname><given-names>PH</given-names></name><name><surname>Raventós</surname><given-names>DS</given-names></name><name><surname>Neve</surname><given-names>S</given-names></name><name><surname>Ravn</surname><given-names>B</given-names></name><name><surname>Bonvin</surname><given-names>AM</given-names></name><name><surname>De Maria</surname><given-names>L</given-names></name><name><surname>Andersen</surname><given-names>AS</given-names></name><name><surname>Gammelgaard</surname><given-names>LK</given-names></name><name><surname>Sahl</surname><given-names>HG</given-names></name><name><surname>Kristensen</surname><given-names>HH</given-names></name></person-group><year>2010</year><article-title>Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II</article-title><source>Science</source><volume>328</volume><fpage>1168</fpage><lpage>1172</lpage><pub-id pub-id-type="doi">10.1126/science.1185723</pub-id></element-citation></ref><ref id="bib55"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schweizer</surname><given-names>F</given-names></name></person-group><year>2009</year><article-title>Cationic amphiphilic peptides with cancer-selective toxicity</article-title><source>European Journal of Pharmacology</source><volume>625</volume><fpage>190</fpage><lpage>194</lpage><pub-id pub-id-type="doi">10.1016/j.ejphar.2009.08.043</pub-id></element-citation></ref><ref id="bib56"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sheetz</surname><given-names>MP</given-names></name></person-group><year>2001</year><article-title>Cell control by membrane-cytoskeleton adhesion</article-title><source>Nature Review Molecular Cell Biology</source><volume>2</volume><fpage>392</fpage><lpage>396</lpage><pub-id pub-id-type="doi">10.1038/35073095</pub-id></element-citation></ref><ref id="bib57"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sheetz</surname><given-names>MP</given-names></name><name><surname>Sable</surname><given-names>JE</given-names></name><name><surname>Dobereiner</surname><given-names>HG</given-names></name></person-group><year>2006</year><article-title>Continuous membrane-cytoskeleton adhesion requires continuous accommodation to lipid and cytoskeleton dynamics</article-title><source>Annual Review of Biophysics and Biomolecular Structure</source><volume>35</volume><fpage>417</fpage><lpage>434</lpage><pub-id pub-id-type="doi">10.1146/annurev.biophys.35.040405.102017</pub-id></element-citation></ref><ref id="bib58"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sok</surname><given-names>M</given-names></name><name><surname>Sentjurc</surname><given-names>M</given-names></name><name><surname>Schara</surname><given-names>M</given-names></name></person-group><year>1999</year><article-title>Membrane fluidity characteristics of human lung cancer</article-title><source>Cancer Letters</source><volume>139</volume><fpage>215</fpage><lpage>220</lpage><pub-id pub-id-type="doi">10.1016/S0304-3835(99)00044-0</pub-id></element-citation></ref><ref id="bib59"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Storoni</surname><given-names>LC</given-names></name><name><surname>Mccoy</surname><given-names>AJ</given-names></name><name><surname>Read</surname><given-names>RJ</given-names></name></person-group><year>2004</year><article-title>Likelihood-enhanced fast rotation functions</article-title><source>Acta Crystallographica Section D, Biological Crystallography</source><volume>60</volume><fpage>432</fpage><lpage>438</lpage><pub-id pub-id-type="doi">10.1107/S0907444903028956</pub-id></element-citation></ref><ref id="bib60"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thevissen</surname><given-names>K</given-names></name><name><surname>Cammue</surname><given-names>BP</given-names></name><name><surname>Lemaire</surname><given-names>K</given-names></name><name><surname>Winderickx</surname><given-names>J</given-names></name><name><surname>Dickson</surname><given-names>RC</given-names></name><name><surname>Lester</surname><given-names>RL</given-names></name><name><surname>Ferket</surname><given-names>KK</given-names></name><name><surname>Van Even</surname><given-names>F</given-names></name><name><surname>Parret</surname><given-names>AH</given-names></name><name><surname>Broekaert</surname><given-names>WF</given-names></name></person-group><year>2000</year><article-title>A gene encoding a sphingolipid biosynthesis enzyme determines the sensitivity of <italic>Saccharomyces cerevisiae</italic> to an antifungal plant defensin from dahlia (<italic>Dahlia merckii</italic>)</article-title><source>Proceedings of the National Academy of Sciences of the United States of America</source><volume>97</volume><fpage>9531</fpage><lpage>9536</lpage><pub-id pub-id-type="doi">10.1073/pnas.160077797</pub-id></element-citation></ref><ref id="bib61"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thevissen</surname><given-names>K</given-names></name><name><surname>Francois</surname><given-names>IE</given-names></name><name><surname>Takemoto</surname><given-names>JY</given-names></name><name><surname>Ferket</surname><given-names>KK</given-names></name><name><surname>Meert</surname><given-names>EM</given-names></name><name><surname>Cammue</surname><given-names>BP</given-names></name></person-group><year>2003</year><article-title>DmAMP1, an antifungal plant defensin from dahlia (<italic>Dahlia merckii</italic>), interacts with sphingolipids from <italic>Saccharomyces cerevisiae</italic></article-title><source>FEMS Microbiology Letters</source><volume>226</volume><fpage>169</fpage><lpage>173</lpage><pub-id pub-id-type="doi">10.1016/S0378-1097(03)00590-1</pub-id></element-citation></ref><ref id="bib62"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thevissen</surname><given-names>K</given-names></name><name><surname>Warnecke</surname><given-names>DC</given-names></name><name><surname>Francois</surname><given-names>IE</given-names></name><name><surname>Leipelt</surname><given-names>M</given-names></name><name><surname>Heinz</surname><given-names>E</given-names></name><name><surname>Ott</surname><given-names>C</given-names></name><name><surname>Zähringer</surname><given-names>U</given-names></name><name><surname>Thomma</surname><given-names>BP</given-names></name><name><surname>Ferket</surname><given-names>KK</given-names></name><name><surname>Cammue</surname><given-names>BP</given-names></name></person-group><year>2004</year><article-title>Defensins from insects and plants interact with fungal glucosylceramides</article-title><source>The Journal of Biological Chemistry</source><volume>279</volume><fpage>3900</fpage><lpage>3905</lpage><pub-id pub-id-type="doi">10.1074/jbc.M311165200</pub-id></element-citation></ref><ref id="bib63"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thevissen</surname><given-names>K</given-names></name><name><surname>Idkowiak-Baldys</surname><given-names>J</given-names></name><name><surname>Im</surname><given-names>YJ</given-names></name><name><surname>Takemoto</surname><given-names>J</given-names></name><name><surname>Francois</surname><given-names>IE</given-names></name><name><surname>Ferket</surname><given-names>KK</given-names></name><name><surname>Aerts</surname><given-names>AM</given-names></name><name><surname>Meert</surname><given-names>EM</given-names></name><name><surname>Winderickx</surname><given-names>J</given-names></name><name><surname>Roosen</surname><given-names>J</given-names></name><name><surname>Cammue</surname><given-names>BP</given-names></name></person-group><year>2005</year><article-title>SKN1, a novel plant defensin-sensitivity gene in <italic>Saccharomyces cerevisiae</italic>, is implicated in sphingolipid biosynthesis</article-title><source>FEBS Letters</source><volume>579</volume><fpage>1973</fpage><lpage>1977</lpage><pub-id pub-id-type="doi">10.1016/j.febslet.2005.02.043</pub-id></element-citation></ref><ref id="bib64"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thomma</surname><given-names>BP</given-names></name><name><surname>Cammue</surname><given-names>BP</given-names></name><name><surname>Thevissen</surname><given-names>K</given-names></name></person-group><year>2003</year><article-title>Mode of action of plant defensins suggests therapeutic potential</article-title><source>Current Drug Targets Infectious Disorders</source><volume>3</volume><fpage>1</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.2174/1568005033342000</pub-id></element-citation></ref><ref id="bib65"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Utsugi</surname><given-names>T</given-names></name><name><surname>Schroit</surname><given-names>AJ</given-names></name><name><surname>Connor</surname><given-names>J</given-names></name><name><surname>Bucana</surname><given-names>CD</given-names></name><name><surname>Fidler</surname><given-names>IJ</given-names></name></person-group><year>1991</year><article-title>Elevated expression of phosphatidylserine in the outer-membrane leaflet of human tumor-cells and recognition by activated human blood monocytes</article-title><source>Cancer Research</source><volume>51</volume><fpage>3062</fpage><lpage>3066</lpage></element-citation></ref><ref id="bib66"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Van der Weerden</surname><given-names>NL</given-names></name><name><surname>Anderson</surname><given-names>MA</given-names></name></person-group><year>2013</year><article-title>Plant defensins: common fold, multiple functions</article-title><source>Fungal Biology Reviews</source><volume>26</volume><fpage>121</fpage><lpage>131</lpage></element-citation></ref><ref id="bib67"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Van der Weerden</surname><given-names>NL</given-names></name><name><surname>Hancock</surname><given-names>RE</given-names></name><name><surname>Anderson</surname><given-names>MA</given-names></name></person-group><year>2010</year><article-title>Permeabilization of fungal hyphae by the plant defensin NaD1 occurs through a cell wall-dependent process</article-title><source>The Journal of Biological Chemistry</source><volume>285</volume><fpage>37513</fpage><lpage>37520</lpage><pub-id pub-id-type="doi">10.1074/jbc.M110.134882</pub-id></element-citation></ref><ref id="bib68"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Van der Weerden</surname><given-names>NL</given-names></name><name><surname>Lay</surname><given-names>FT</given-names></name><name><surname>Anderson</surname><given-names>MA</given-names></name></person-group><year>2008</year><article-title>The plant defensin, NaD1, enters the cytoplasm of <italic>Fusarium oxysporum</italic> hyphae</article-title><source>The Journal of Biological Chemistry</source><volume>283</volume><fpage>14445</fpage><lpage>14452</lpage><pub-id pub-id-type="doi">10.1074/jbc.M709867200</pub-id></element-citation></ref><ref id="bib69"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Van Meer</surname><given-names>G</given-names></name><name><surname>Voelker</surname><given-names>DR</given-names></name><name><surname>Feigenson</surname><given-names>GW</given-names></name></person-group><year>2008</year><article-title>Membrane lipids: where they are and how they behave</article-title><source>Nature Review Molecular Cell Biology</source><volume>9</volume><fpage>112</fpage><lpage>124</lpage><pub-id pub-id-type="doi">10.1038/nrm2330</pub-id></element-citation></ref><ref id="bib70"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wei</surname><given-names>G</given-names></name><name><surname>Pazgier</surname><given-names>M</given-names></name><name><surname>De Leeuw</surname><given-names>E</given-names></name><name><surname>Rajabi</surname><given-names>M</given-names></name><name><surname>Li</surname><given-names>J</given-names></name><name><surname>Zou</surname><given-names>G</given-names></name><name><surname>Jung</surname><given-names>G</given-names></name><name><surname>Yuan</surname><given-names>W</given-names></name><name><surname>Lu</surname><given-names>WY</given-names></name><name><surname>Lehrer</surname><given-names>RI</given-names></name><name><surname>Lu</surname><given-names>W</given-names></name></person-group><year>2010</year><article-title>Trp-26 imparts functional versatility to human alpha-defensin HNP1</article-title><source>The Journal of Biological Chemistry</source><volume>285</volume><fpage>16275</fpage><lpage>16285</lpage><pub-id pub-id-type="doi">10.1074/jbc.M110.102749</pub-id></element-citation></ref><ref id="bib71"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wiedemann</surname><given-names>I</given-names></name><name><surname>Breukink</surname><given-names>E</given-names></name><name><surname>Van Kraaij</surname><given-names>C</given-names></name><name><surname>Kuipers</surname><given-names>OP</given-names></name><name><surname>Bierbaum</surname><given-names>G</given-names></name><name><surname>De Kruijff</surname><given-names>B</given-names></name><name><surname>Sahl</surname><given-names>HG</given-names></name></person-group><year>2001</year><article-title>Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity</article-title><source>The Journal of Biological Chemistry</source><volume>276</volume><fpage>1772</fpage><lpage>1779</lpage><pub-id pub-id-type="doi">10.1074/jbc.M006770200</pub-id></element-citation></ref><ref id="bib72"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wilmes</surname><given-names>M</given-names></name><name><surname>Cammue</surname><given-names>BPA</given-names></name><name><surname>Sahl</surname><given-names>HG</given-names></name><name><surname>Thevissen</surname><given-names>K</given-names></name></person-group><year>2011</year><article-title>Antibiotic activities of host defense peptides: more to it than lipid bilayer perturbation</article-title><source>Natural Product Reports</source><volume>28</volume><fpage>1350</fpage><lpage>1358</lpage><pub-id pub-id-type="doi">10.1039/c1np00022e</pub-id></element-citation></ref><ref id="bib73"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wimley</surname><given-names>WC</given-names></name><name><surname>Selsted</surname><given-names>ME</given-names></name><name><surname>White</surname><given-names>SH</given-names></name></person-group><year>1994</year><article-title>Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores</article-title><source>Protein Science</source><volume>3</volume><fpage>1362</fpage><lpage>1373</lpage><pub-id pub-id-type="doi">10.1002/pro.5560030902</pub-id></element-citation></ref><ref id="bib74"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yoon</surname><given-names>WH</given-names></name><name><surname>Park</surname><given-names>HD</given-names></name><name><surname>Lim</surname><given-names>K</given-names></name><name><surname>Hwang</surname><given-names>BD</given-names></name></person-group><year>1996</year><article-title>Effect of O-glycosylated mucin on invasion and metastasis of HM7 human colon cancer cells</article-title><source>Biochemical and Biophysical Research</source><volume>222</volume><fpage>694</fpage><lpage>699</lpage><pub-id pub-id-type="doi">10.1006/bbrc.1996.0806</pub-id></element-citation></ref><ref id="bib75"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zeisig</surname><given-names>R</given-names></name><name><surname>Koklic</surname><given-names>T</given-names></name><name><surname>Wiesner</surname><given-names>B</given-names></name><name><surname>Fichtner</surname><given-names>I</given-names></name><name><surname>Sentjurc</surname><given-names>M</given-names></name></person-group><year>2007</year><article-title>Increase in fluidity in the membrane of MT3 breast cancer cells correlates with enhanced cell adhesion in vitro and increased lung metastasis in NOD/SCID mice</article-title><source>Archives of Biochemistry and Biophysics</source><volume>459</volume><fpage>98</fpage><lpage>106</lpage><pub-id pub-id-type="doi">10.1016/j.abb.2006.09.030</pub-id></element-citation></ref><ref id="bib76"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Rozek</surname><given-names>A</given-names></name><name><surname>Hancock</surname><given-names>RE</given-names></name></person-group><year>2001</year><article-title>Interaction of cationic antimicrobial peptides with model membranes</article-title><source>The Journal of Biological Chemistry</source><volume>276</volume><fpage>35714</fpage><lpage>35722</lpage><pub-id pub-id-type="doi">10.1074/jbc.M104925200</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.01808.033</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://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 “PtdIns(4,5)P<sub>2</sub>-mediated oligomerization of the defensin NaD1 represents a novel mechanism of cell lysis” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor, John Kuriyan, and 2 reviewers.</p><p>The editor and the other reviewers discussed their comments before we reached this decision, and the editor has assembled the following comments to help you prepare a revised submission.</p><p>The manuscript by Poon et al. describes a thorough structure-function study of the ornamental tobacco defensin NaD1. Defensins belong to the group of cationic antimicrobial peptides, which function as ubiquitously expressed innate immune molecules that often destabilize membrane structure as the detrimental step for the target cells. While plant defensins share a common structure, their primary sequences are less conserved, which is likely linked to their apparent ligand specificity. In previous work, this research team determined the basic structure and cellular mode of action of NaD1. Here, they describe several new discoveries that present a large step forward in our molecular understanding of these peptides. In particular, this is the first report of any defensin making a specific interaction with a phosphoinositide, and the structural information provided by the work is really first-rate and informative.</p><p>The authors identify the membrane lipid PIP<sub>2</sub> as the high-affinity ligand for NaD1 in fungal and mammalian cells. Furthermore, they demonstrate, based on a novel crystal structure and biochemical data, that NaD1 oligomerizes upon PIP<sub>2</sub> binding and this step significantly increases NaD1's cytolytic activity. While the PIP<sub>2</sub> binding sites created by NaD1 oligomers has features seen in other PIP<sub>2</sub> binding modules, NaD1 adopts a fold distinct from the other well-established protein domains. Finally, elegant experiments establish the timing of events triggered by NaD1, in particular membrane blebbing, cell permeabilization and cytolysis. Taken together, this is a well-written and executed study that makes a significant contribution to the field. Given the broad expression of defensins and the general importance of PIP2 for cellular physiology, this work should be of great interest to the diverse readership of <italic>eLife</italic>.</p><p>The reviewers have identified one issue that should perhaps be addressed experimentally before submitting a revised paper, and hopefully this should cause little difficulty. The revised manuscript will be considered by the editor without further input from the reviewers, and so a clear description of what is done in the revision would help make a quick decision.</p><p>Important experimental issue:</p><p>The discussion on PIP specificity relies heavily on the protein-overlay assays that utilize PIP Strips, which are notoriously unreliable. The manner in which the lipids are displayed on these blots bears little resemblance to how they are displayed in lipid bilayers. The authors have presented single blots and their quantitation is simply densitometry of these single strips. The authors should give the readers a realistic indication as to how reproducible these blots are. There should be errors indicated on the quantitation that refer to biological replicates. These strips can vary wildly from one experiment to another and from batch to batch (they were commercially supplied). Ideally, it would be helpful if the authors used measurements on liposomes, but this is not essential. But, unless validated by liposome binding experiments, the authors should refrain from quantitative arguments. Along these lines, quantification of NaD1 binding (wild type vs mutant) to PIP-containing liposomes is also desirable since it may reveal significant differences that are not apparent in the more artificial PIP Strip assays.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01808.034</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>The discussion on PIP specificity relies heavily on the protein-overlay assays that utilize PIP Strips, which are notoriously unreliable. The manner in which the lipids are displayed on these blots bears little resemblance to how they are displayed in lipid bilayers. The authors have presented single blots and their quantitation is simply densitometry of these single strips. The authors should give the readers a realistic indication as to how reproducible these blots are. There should be errors indicated on the quantitation that refer to biological replicates. These strips can vary wildly from one experiment to another and from batch to batch (they were commercially supplied). Ideally, it would be helpful if the authors used measurements on liposomes, but this is not essential. But, unless validated by liposome binding experiments, the authors should refrain from quantitative arguments. Along these lines, quantification of NaD1 binding (wild type vs mutant) to PIP-containing liposomes is also desirable since it may reveal significant differences that are not apparent in the more artificial PIP Strip assays</italic>.</p><p>The reviewers raise an excellent point for the protein overlay lipid strips in that we should provide an indication of reproducibility by including errors on the quantitation referring to biological replicates. We have performed statistical analysis on biological replicates for the relevant experiments, and have amended <xref ref-type="fig" rid="fig1s1">Figure 1–figure supplement 1</xref> and <xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1</xref>, accordingly.</p><p>The reviewers also suggested that it would be helpful (but not essential) to use liposome-binding experiments to quantify NaD1 binding (wild-type vs mutant). We agree that these data would provide support to the lipid-blot data and have attempted the experiments. However, we were not able to adequately resolve a number of technical challenges in a reasonable time frame, which was exacerbated by the subsequent need for the production of additional recombinant proteins. The reviewers suggested that if were unable to provide quantitative liposome pull-down data, we should refrain from quantitative arguments. As such, we have modified the text accordingly by replacing several statements, as follows:</p><p>A) In the first Results paragraph “Noticeably, NaD1 bound strongest to PIP2 relative to all other lipids tested....”, has been changed to “Interestingly, NaD1 bound the functionally important phospholipid PIP2....”</p><p>B) In the Results section entitled “PIP2 binding and oligomerization of NaD1 are critical for fungal cell killing”, “...resulted in a dramatic reduction in the binding of PI(4)P” has been replaced with “However, it did result in reduced binding of PI(4)P...”</p></body></sub-article></article> |