While most of the drugs available in the market are competitive inhibitors, there is a rapidly growing interest in development of allosteric drugs, particularly to inhibit protein-protein interactions (PPI) with large interaction surface area. However, it remains a challenge to identify a distal binding site that would be allosterically linked to the canonical ligand/substrate binding site. Such allosteric hotspots are often cryptic sites with a less populated excited conformational state of the protein. In this work we present a general strategy based on thermodynamic arguments to identify such distal cryptic sites as potential targets for allosteric drugs. We demonstrate this on allosterically modulating the PPI between PCSK9 (proprotein convertase subtilisin/Kexin type 9) and LDLR (low density lipoprotein receptor), which is a challenging and therapeutically important target towards treatment of hypercholesterolemia (elevated plasma level of LDL). Using several µs long molecular dynamics (MD) simulations, we demonstrate that on binding with the EGF-A domain of LDLR, there is a significant conformational change (population shift) in a distal loop (residues 211-222) region of PCSK9. We have identified several (meta)stable and kinetically resolved conformational states of this loop and demonstrated that there exists a clear correlation between the loop conformation and the binding affinity with LDLR. Using a thermodynamic argument, we establish that the loop conformations predominantly present in the apo state of PCSK9 would have lower binding affinity with LDLR and they would be potential targets for designing allosteric inhibitors. We also elucidate the molecular origin of the allosteric coupling between this loop and PCSK9-LDLR binding interface in terms of population shift in several specific pair-wise interactions consisting of salt bridges and hydrogen bonds. Overall, our work provides a general strategy towards identifying allosteric hotspots, where one should compare the conformational ensemble between the apo and substrate bound states of the protein and identify distal differences, if any. Subsequently the apo-like conformations should be targeted for designing inhibitors that would specifically bind to those conformations and stabilise them.
Computational details related to system setup and simulation methods, additional analysis results supporting the observations reported here.