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. 2015 Dec 29;112(52):15910-5.
doi: 10.1073/pnas.1518946112. Epub 2015 Dec 11.

Detection of secondary binding sites in proteins using fragment screening

R Frederick Ludlow  1 , Marcel L Verdonk  1 , Harpreet K Saini  1 , Ian J Tickle  1 , Harren Jhoti  2
Affiliations

Detection of secondary binding sites in proteins using fragment screening

R Frederick Ludlow et al. Proc Natl Acad Sci U S A. .

Abstract

Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

Keywords: X-ray crystallography; fragment-based drug design; protein function; protein structure.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Overlaid binding sites observed for four targets in this study. (A) CDK2 (PDB 5FP5) with compound 1 (yellow, cyan, PDB 5FP5) and compound 2 (green, PDB 5FP6). (Inset) Overlay of d-Luceferin derivative from PDB 4D1Z and compound 1. (B) HSPA2 protein from PDB 5FPE with fragments 3 (dark gray and cyan), 4 (green, PDB 5FPM), 5 (light gray, PDB 5FPD), and 6 (orange, PDB 5FPN). (C) DNA ligase PDB 5FPO with fragment 7 (green, PDB 5FPR) and 8 (yellow, cyan, PDB 5FPO). (D) Protease (gray) and helicase (blue) domains of HCV NS3 in complex with 8 (gray, PDB 4B6E), 9 (green, PDB 5FPS), 10 (yellow, PDB 5FPY), 11 (cyan, PDB 5FPT). (Inset) Overlay of our fragment 11 (cyan, PDB 5FPT) with compound 12 reported by Boehringer Ingelheim (yellow, PDB 4OJQ).
Fig. 2.
Fig. 2.
Ortholog site vs. global sequence identity for (A) primary sites (active sites, known cofactors, etc.) and (B) secondary sites. Each point represents a single ortholog sequence, jitter applied for clarity. A reference plot (the distribution expected if there was no particular conservation of the site residues) is shown in Supporting Information (Fig. S1).
Fig. S1.
Fig. S1.
Null hypothesis plot for ortholog site vs. global sequence identity. Both primary and secondary sites are combined here. Each point represents a single ortholog sequence, jitter applied for clarity.
Fig. S2.
Fig. S2.
Site sequence identity plotted against global sequence identity for all secondary sites with unknown function.
Fig. S3.
Fig. S3.
Site sequence identity plotted against global sequence identity for the five fragment binding sites in HSPA2. Site 1: ATP site, compound 3 (dark gray fragment, PDB 5FPE); 2: cyan fragment, compound 3, PDB 5FPE; 3: compound 4, green fragment, PDB 5FPM; 4: compound 5, light gray, PDB 5FPD; and 5: compound 6, orange, PDB 5FPN.
Fig. 3.
Fig. 3.
Flexibility of primary (green)- and secondary (red)-site atoms, compared with general protein surface atoms (blue). Results are shown for (A) the mean computed local atomic mobility index (AMI) for the structures in our test set, and (B) the mean normalized B factors as observed in X-ray apo structures. Sites for which we developed a potent lead compound are shown in filled circles. The empty circles are sites we have not attempted to, or were so far unable to develop potent lead compounds against.
Fig. S4.
Fig. S4.
Normalized B factors of all surface atoms for each target (blue) and the subset of surface atoms in primary and secondary sites (green and red, respectively).
Fig. S5.
Fig. S5.
Fraction of the ligand area that is buried on binding to the protein (Left) and fraction of the protein area contacting the ligand that is polar (Right). Results are shown for the primary (green) and secondary (red) sites that were part of our analysis. Filled circles represent sites against which we have been able to develop potent lead compounds. The horizontal line on the Right represents the average fraction of the total protein surface area that is polar.
Fig. S6.
Fig. S6.
Histograms for primary sites (blue) and secondary sites (red) depicting ligand properties ClogP, heavy-atom count (HAC), number of H-bond donors (NDON), and number of H-bond acceptors (NACC).
See this image and copyright information in PMC

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