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Identifying binding hot spots on protein surfaces by mixed‐solvent molecular dynamics: HIV‐1 protease as a test case

Mixed‐solvent molecular dynamics (MixMD) simulations use full protein flexibility and competition between water and small organic probes to achieve accurate hot‐spot mapping on protein surfaces. In this study, we improved MixMD using human immunodeficiency virus type‐1 protease (HIVp) as the test case. We used three probe–water solutions (acetonitrile–water, isopropanol–water, and pyrimidine–water), first at 50% w/w concentration and later at 5% v/v. Paradoxically, better mapping was achieved by using fewer probes; 5% simulations gave a superior signal‐to‐noise ratio and far fewer spurious hot spots than 50% MixMD. Furthermore, very intense and well‐defined probe occupancies were observed in the catalytic site and potential allosteric sites that have been confirmed experimentally. The Eye site, an allosteric site underneath the flap of HIVp, has been confirmed by the presence of a 5‐nitroindole fragment in a crystal structure. MixMD also mapped two additional hot spots: the Exo site (between the Gly16‐Gly17 and Cys67‐Gly68 loops) and the Face site (between Glu21‐Ala22 and Val84‐Ile85 loops). The Exo site was observed to overlap with crystallographic additives such as acetate and dimethyl sulfoxide that are present in different crystal forms of the protein. Analysis of crystal structures of HIVp in different symmetry groups has shown that some surface sites are common interfaces for crystal contacts, which means that they are surfaces that are relatively easy to desolvate and complement with organic molecules. MixMD should identify these sites; in fact, their occupancy values help establish a solid cut‐off where “druggable” sites are required to have higher occupancies than the crystal‐packing faces. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 21–34, 2016.