Multidrug resistance to current FDA approved HIV-1 protease (PR) inhibitors drives the need to understand the fundamental mechanisms of how drug-pressure selected mutations, which are oftentimes natural polymorphisms, elicit their effect on enzyme function and resistance. Here, the impact of hinge-region natural polymorphism at residue 35 – glutamate to aspartate (E35D) – alone and in conjunction with residue 57 arginine to lysine (R57K) are characterized with the goal of understanding how altered salt-bridge interactions between the hinge and flap regions are associated with changes in structure, motional dynamics, conformational sampling, kinetic parameters, and inhibitor affinity. The combined results reveal that the single E35D substitution leads to diminished salt-bridge interactions between residue 35 and 57 and gives rise to the stabilization of open-like conformational states with overall increased backbone dynamics. In HIV-1 PR constructs where sites 35 and 57 are both mutated (e.g. E35D, R57K), X-ray structures reveal an altered network of interactions that replace the salt-bridge thus stabilizing the structural integrity between the flap and hinge regions. In spite of the altered conformational sampling and dynamics when the salt bridge is disrupted, enzyme kinetic parameters and inhibition constants are similar to those obtained for subtype B PR. Results demonstrate that these hinge region natural polymorphisms, which may arise as drug pressure secondary mutations, alter protein dynamics and conformational landscape, which are important thermodynamic parameters to consider for development of inhibitors that target for non-subtype B PR.
Effects of Hinge Region Natural Polymorphisms on Human Immunodeficiency Virus-1 Protease Structure, Dynamics and Drug-Pressure Evolution.