Site mining - Binding site characterization

 Site mining with MED-SuMo: searching, browsing and superposing 3D macromolecules structures

Superposing protein active sites of a given superfamily (protein phosphatase, protein kinases, serine proteases ...) helps to deciphering the contributions to affinity and selectivity of the protein's surface chemical features. This binding site characterization illuminates:

   Conformational diversity

   Functionnal diversity

The method applies to all protein superfamilies highly represented in the PDB like the protein kinases, serine proteases, phosphodiesterases, HSP90, beta secretase, protein phosphatase...

 Searching the PDB with a PDE4 binding site

In this application, MED-SuMo is used to explore the difference between two phosphodiesterases PDE4 and PDE5. Their catalytic domain are highly conserved, especially for PDE4 and PDE5 which is not surprising since they address very close ligand: cAMP and cGMP.

The MED-SuMo Graphical User interface helps to compare one PDE structure to the whole PDB. to view superpositions and to analyse the signatures of the binding sites (Fig.1).

Fig.1: Binding site characterization application within MED-SuMo GUI 1.1.38. A protein phosphatase is used as a query (PDB code 2VEW). The result table is shown in the clustering mode: the hits are classified according to the surface chemical features shared with the query. The consensus signature is shown for each of the 13 clusters. In the 3D viewer, one hit from each cluster is displayed n the 3D viewer (2I75, 2HY3, 1NZ7, 1YTS, 1XXP, 1D5R, 1AAX, 1OHD, 2I42). Actually only 9 among the 13 clusters contain only true positives.

 Analysis of the results: higlighting the importance of rotamers of GLN 443 residue

In this application, we've compared the PDE4-cAMP complexe (PDB code 1ROR) to the whole PDB. The first hit, which is a PDE5, is 1T9S and is ranked 36th just after many PDE4 and 2 PDE10 (click on the image on the left to enlarge). The score of 1T9S, is high (18.2) and corresponds to 65% of the maximum score achievable (exact match to the query). This indicates that PDE4 and PDE5 share most of their active site. The main difference is found at the Tyr403, which is a different amino acid in the PDE5 and is not directly involved in the interaction with the ligand.

Another interesting point is the absence, for PDE5, of the Surface Chemical Features which represents the amide function of the Gln443 of PDE4 (This SCF is green). It is known that in the PDE4-cAMP interaction the nucleotide is recognized by a bidentate H-bond motif involving Gln443, a residue conserved across all PDE families. The rotation of this critical amide side chain is fixed by a network of additional H bonds.

The recognition of a guanine (as in cGMP) instead of adenine (in the cAMP), as in cGMP-specific PDE5, goes with the rotation which reverses the amide side chain (inside the yellow ellipse, figure on the left) allowing to meet the inverted H-bond of the lactam of guanine. If we look at the 3D superimposition of 1ROR and 1T9S we can see that MED-SuMo is able to detect this structural difference between PDE4 and PDE5 (on the left). There isn’t an amide SuMo object in the PDE5 signature because SuMo detects the different position of the Gln side chain. MED-SuMo characterizes in term of chemical function in 3D the active site of the PDE.

Fig.2 View of the superimposition of 1ROR-AMP (grey) and 1T9S-GMP (green; proteins and ligands are represented). Inside the yellow ellipse, the reverse position of the amide group of the Gln without any amide SuMo-object can be seen.

To know if this difference between PDE4 and PDE5 is always conserved with different ligands, a PDE5 complexed with vardenafil is used as a second query (PDB code: 1XP0) towards the whole PDB. The results show that the hits at the top of the list are all PDE5 followed by PDE4 (click on the image on the left to enlarge). It can be seen  that the SuMo object which represents the amide function of the Gln817 in the query is not present in the signatures of the PDE4s. It can also be seen in the result table that the PDE5 structure 2H44 misses the GLN817 amide object as well, the visualisation of the superposition indicates that they are rotated by about 90 degrees.

 MED-SuMo database

The MED-SuMo server parameters are sharply optimized around default values, to get the highest quality results. For this binding site characterization application, we are seeking for similar binding sites to a given binding site and we are looking precisely at chemical features:

     Site database

       In this case study, we are seeking for similar binding sites to a given binding site.

     Binding site description with a high number of triplets, the Best mode

       The focus is on the surface chemical features.

   Shape threshold = 45%:

      The shape similarity cutoff is lower to optimize the probability of hitting related binding sites without introducting false positives.

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