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MurE fragment screen

Chris Swain edited this page May 8, 2019 · 52 revisions

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.

MurEsubstrate

Updated with additional crystal structures

The crystal structure contains two copies of the protein in the asymmetric unit. The fragments bind in 17 different positions on the protein, 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

Clusters 1 to 4

MUREECA-x0062_1
MUREECA-x0062_2
MUREECA-x0062_3
MUREECA-x0062_4

Cluster1

The same fragment is binding at different sites remote from the active site.

Cluster 5

MUREECA-x0100_1

A single fragment binding at a position remote from the active site.

Cluster5

Cluster 6

MUREECA-x0114_1
MUREECA-x0198_3

This contains two similar fragments, and they bind at a point very close to the substrate binding site.

Cluster2

Looking at the binding site for the cluster 2 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.

MissingLoop

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.

substrate binding

Binding at this site is more likely to have an influence on enzyme inhibition.

Cluster 7

This contains two fragments, this is the same binding site as Cluster 6 but on the other monomer of the asymmetric unit.

Cluster 8, 9 & 10

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.

cluster4_5
clusters4_5

Cluster 11

MUREECA-x0175_1

Cluster11

Cluster 12

MUREECA-x0197_1

Cluster12

Clusters 13 and 14

This contains two fragments

Cluster9

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.

Cluster9 site

Cluster 15

MUREECA-x0206_1
MUREECA-x0209_1

This contains two fragments

MurECluster15

These fragments occupy the ADP site.The substrate is shown in green, and ADP in blue, and the fragment in purple.

Cluster15 site

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.

MurE0206_1bindingInteractions

You can view and rotate the 3D structure of the enzyme with fragment bound here.

Cluster 16

MUREECA-x0206_2

This contains only a single fragment.

Cluster 17

MUREECA-x0215_2

This contains only a single fragment

MurECluster17

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.

Cluster17 site
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