Kinetic, Stability, and Structural Changes in High-resolution Crystal Structures of HIV-1 Protease with Drug-resistant Mutations L24I, I50V, and G73S

The crystal structures, dimer stabilities, and kinetics have been analyzed for wild-type human immunodeficiency virus type 1 (HIV-1) protease (PR) and resistant mutants PRL24I, PRI50V, and PRG73S to gain insight into the molecular basis of drug resistance. The mutations lie in different structural regions. Mutation I50V alters a residue in the flexible flap that interacts with the inhibitor, L24I alters a residue adjacent to the catalytic Asp25, and G73S lies at the protein surface far from the inhibitor-binding site. PRL24I and PRI50V, showed a 4% and 18% lower kcat/Km, respectively, relative to PR. The relative kcat/Km of PRG73S varied from 14% to 400% when assayed using different substrates. Inhibition constants (Ki) of the antiviral drug indinavir for the reaction catalyzed by the mutant enzymes were about threefold and 50-fold higher for PRL24I and PRI50V, respectively, relative to PR and PRG73S. The dimer dissociation constant (Kd) was estimated to be approximately 20 nM for both PRL24I and PRI50V, and below 5 nM for PRG73S and PR. Crystal structures of the mutants PRL24I, PRI50V and PRG73S were determined in complexes with indinavir, or the p2/NC substrate analog at resolutions of 1.10–1.50 Å. Each mutant revealed distinct structural changes relative to PR. The mutated residues in PRL24I and PRI50V had reduced intersubunit contacts, consistent with the increased Kd for dimer dissociation. Relative to PR, PRI50V had fewer interactions of Val50 with inhibitors, in agreement with the dramatically increased Ki. The distal mutation G73S introduced new hydrogen bond interactions that can transmit changes to the substrate-binding site and alter catalytic activity. Therefore, the structural alterations observed for drug-resistant mutations were in agreement with kinetic and stability changes.