Initial MurE Hits
The fragment screen was run against apo MurE ligase from Escherichia coli Uniprot P22188, the X-ray screen was carried out at Diamond and identified a number of hits described below.
The crystal structure of MurE ligase from Escherichia coli has been published PDB code 1E8C with the substrate uridine-5'-diphosphate-n-acetylmuramoyl-l-alanine-d-glutamate bound in the active site. The structure of MurE from M.tuberculosis with dipeptide (shown below in green) and ADP (shown below in purple)bound has also been published PDB code 2XJA.
The crystal structure contains two copies of the protein in the asymmetric unit. The fragments bind in 17 different positions on the protein (shown in the image below), in some cases there is only a single fragment bound at a position, in other cases there are clusters of fragments binding in a similar region.
The Fragment numbers refer to the unique crystal number assigned at Diamond. https://fragalysis.diamond.ac.uk/viewer/react/preview/target/MUREECA
MUREECA-x0062_1
MUREECA-x0062_2
MUREECA-x0062_3
MUREECA-x0062_4
The same fragment is binding at different sites remote from the active site.
MUREECA-x0100_1
A single fragment binding at a position remote from the active site.
MUREECA-x0114_1
MUREECA-x0198_3
This contains two similar fragments, and they bind at a point very close to the substrate binding site.
Looking at the binding site for the cluster 6 fragments in more detail and comparing it with the protein with substrate bound gives a better insight into an important detail. The fragments are shown in purple and the substrate in light green in the image below. In the crystal structure of the fragment bound to the protein a loop is missing, this usually suggests that it is disordered. It has now been confirmed that this loop is disordered. The two ends of the missing loop are highlighted by the orange arrows. The corresponding loop in the protein with substrate bound is shown in dark green, as can be seen this loop passes through the region occupied by the fragments.
The importance of this loop can be seen in the image below showing some of the key interactions between substrate and protein. The dark green loop contains 3 residues (Asn156, Thr157, Thr158) which contribute to substrate binding.
Binding at this site is more likely to have an influence on enzyme inhibition.
This contains two fragments, this is the same binding site as Cluster 6 but on the other monomer of the asymmetric unit.
MUREECA-x0164_1
MUREECA-x0237_3
MUREECA-x0172_3
MUREECA-x0237_2
Clusters 8, 9 and 10 lie at the crystal interface between the monomers, each is the same single molecule. This is probably an artefact of the crystal packing.
MUREECA-x0175_1
MUREECA-x0197_1
This contains two fragments
However again this site is remote from the active site.The substrate is shown in green, and ADP in blue, and the fragment in purple.
MUREECA-x0206_1
MUREECA-x0209_1
This contains two fragments
These fragments occupy the ADP site.The substrate is shown in green, and ADP in blue, and the fragment in purple.
The pyrimidine ring sits between Phe307 and Asn118 with one of the pyrimidine nitrogens hydrogen bonding to Lys367. The secondary alcohol makes hydrogen bonds to Ser310 and Asn311.
You can view and rotate the 3D structure of the enzyme with fragment bound here.
MUREECA-x0206_2
This contains only a single fragment.
MUREECA-x0215_2
This contains only a single fragment
However again this site is remote from the active site.The substrate is shown in green, and ADP in blue, and the fragment in purple.
You can now view the Cluster 6 and Cluster 15 fragments using NGL viewer here
You can view the structures of non-binders here
Some follow up work on Clusters 6 and 15 has been done and is described here.