Anyone who has been exposed to much crystallography will have seen examples where a ligand binds somewhere besides the active site of a protein. This is probably all the more likely in the case of fragments, both because fragments are soaked at high concentrations (and thus weaker ligands can be detected) and also because, being less complex, fragments will be able to bind to more sites. In some cases, such as FPPS and HCV NS3, ligands that bind at these “secondary sites” could be advanced to potent allosteric inhibitors. But how common are such sites? This is the question addressed by Harren Jhoti and Astex colleagues in a paper just published in Proc. Nat. Acad. Sci USA.
The researchers were privileged to have 5590 crystal structures of 24 proteins with at least one bound ligand from crystallographic fragment screens. Careful analysis to exclude buffers and molecules bound at crystallograpic interfaces left them with 53 sites total, with each protein having a fragment bound in at least 1 site; one had 6 (still far from the record 16 sites in HIV-1 RT discussed here). Importantly, 16 of the targets had at least 2 ligand binding sites, with an overall average of 2.2. This number of secondary sites is likely a lower bound, as some sites may have been blocked by crystal packing.
What can be said about these ligand binding sites? The researchers compared the sequence conservation between orthologous proteins from different organisms and found that primary binding sites are more conserved than the overall protein sequences. This is expected because, since the proteins likely have similar functions, there are more evolutionary constraints on the active site residues surrounding the primary sites. Interestingly though, the secondary sites were also significantly conserved, suggesting that they too may have some sort of function.
Protein mobility was also examined computationally, with the thought being that functional binding sites should be more rigid than the overall surface of the protein so as to minimize entropic costs of ligand binding. This turned out to be the case for all primary ligand binding sites, but it was also true for most of the secondary sites. Surprisingly, and in contrast to previous results, there were no differences in normalized B factors (roughly, temperature-related motions) for residues in either primary or secondary binding sites compared with surface residues in general.
Comparing the physical properties of the primary and secondary sites revealed that both were more lipophilic than the rest of the protein surface. Ligands tended to be slightly more buried in primary binding sites than in secondary sites, but there didn’t seem to be any differences among the ligands themselves, though the twelve shown in the paper are mostly “flat.”
These combined results suggest that the majority of proteins have multiple sites capable of binding to small molecule ligands. The researchers note that most of their examples are enzymes, so it may not be fair to extrapolate to other protein classes. That said, many GPCRs also have multiple ligand binding sites.
Secondary binding sites have several things going for them. First, allosteric sites provide a means to target proteins in which the primary binding site is problematic, perhaps because it is too closely related to other proteins. Allosteric sites can also be useful for targeting viral or cancer targets in which resistance is an issue, as in the case of ABL001. Finally, secondary sites provide an opportunity to develop not just inhibitors, but activators.
Of course, just because a fragment binds at a site doesn’t necessarily mean that the site is ligandable. Indeed, HSP70 appears to have 5 sites, yet by all accounts is an extremely difficult target. Four of the proteins (including HSP70) are described in some detail in the paper, with protein-fragment structures deposited in the protein data bank. It would be interesting to see how the secondary sites score as potential hot spots using software such as FTMap.