Initial MurD Hits
The fragment screen was run against apo MurD ligase from Streptococcus agalactiae Uniprot Q8E186, the X-ray screen was carried out at Diamond and identified 4 hits shown below.
In contrast to the known inhibitors which occupy the UMA binding site the four fragments occupy a pocket remote from the UMA binding site adjacent to site were ADP is bound in the E. coli crystal structures. The image below shows the crystal structures of the four fragments (green) bound to Streptococcus agalactiae MurD ligase. These are overlaid with MurD ligase PDB code 3uag (protein not visible) with associated ADP (coloured blue) and UDP-N-acetylmuramoyl-L-alanine (UMA coloured pink).
It needs to born in mind that since the fragments are binding to a pocket adjacent to the substrate binding site they may not act as inhibitors. The binding may have no functional significance. However it may be possible to build on the fragments towards the substrate binding site. Interestingly this loop (highlighted in pink below) has been suggested as a potential site for allosteric modulators. DOI.
A region which could potentially be targeted by allosteric inhibitors is the hinge loop, which is involved in the C-terminal domain rotation. Thr294, Thr295, His301, Arg302 and Glu304 could be specifically targeted for potential polar interactions with inhibitors due to the presence of several lone electron pairs in their side chains. Structure-based designed allosteric inhibitors with affinity towards this loop could either prevent the binding of substrates or freeze the enzyme in the conformation where domain rotation and consequently catalytic activity are prevented.
The Fragment numbers refer to the unique crystal number assigned at Diamond. https://fragalysis.diamond.ac.uk/viewer/react/preview/target/MURD
Fragment MURD-x0349 makes few binding interactions, the atoms with blue highlighting are solvent exposed with the protonated piperazine nitrogen hydrogen bonding to water. Ile147 sits over the aromatic ring and the nitrile points towards Lys330.
This can be seen more clearly in the site view below.
You can view and rotate the 3D structure of the enzyme with fragment bound here.
Fragment MURD-x0373 makes several important interactions. In this case the protonated piperazine nitrogen interacts with Glu132 with much of the piperazine solvent exposed. The phenyl ring is sandwiched between Ile147 and a methylene of Lys311. The urea carbonyl forms a hydrogen bond with the backbone NH of Lys311.
This can be seen more clearly in the site view below.
You can view and rotate the 3D structure of the enzyme with fragment bound here.
Fragment MURD-x0374 binds in a similar fashion to fragment 373. In this case one of the hydroxyls interacts with Glu132 and the phenyl ring is sandwiched between Ile147 and a methylene of Lys311. The urea carbonyl forms a hydrogen bond with the backbone NH of Lys311. The other hydroxyl and one of the urea NH form hydrogen bonds to waters 337 and 341. There is also a second fragment molecule bound to the surface remote from the site the other fragments bind.
This can be seen more clearly in the site view below.
You can view and rotate the 3D structure of the enzyme with fragment bound here.
Fragment MURD-x0378 binds in a similar fashion the phenyl ring is sandwiched between Ile147 and a methylene of Lys311. The amide carbonyl forms a hydrogen bond with the backbone NH of Lys311.
This can be seen more clearly in the site view below.
You can view and rotate the 3D structure of the enzyme with fragment bound here.
A clearer view of the relative positions of the Fragments and the ADP binding site.
You can now view the fragments using NGL viewer here
You can see just the fragments superimposed here.
Some follow up work on these hit fragments has been done and is described here.