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

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• 作者：Fengling Liu ; Peter I. Boross ; Yuan-Fang Wang ; Jozsef Tozser ; John M. Louis ; Robert W. Harrison and Irene T. Weber
• 刊名：Journal of Molecular Biology
• 出版年：2005
• 出版时间：9 December 2005
• 年：2005
• 卷：354
• 期：4
• 页码：789-800
• 全文大小：685 K

文摘

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 PR_L24I, PR_I50V, and PR_G73S 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. PR_L24I and PR_I50V, showed a 4 % and 18 % lower k_cat/K_m, respectively, relative to PR. The relative k_cat/K_m of PR_G73S varied from 14 % to 400 % when assayed using different substrates. Inhibition constants (K_i) of the antiviral drug indinavir for the reaction catalyzed by the mutant enzymes were about threefold and 50-fold higher for PR_L24I and PR_I50V, respectively, relative to PR and PR_G73S. The dimer dissociation constant (K_d) was estimated to be approximately 20 nM for both PR_L24I and PR_I50V, and below 5 nM for PR_G73S and PR. Crystal structures of the mutants PR_L24I, PR_I50V and PR_G73S 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 PR_L24I and PR_I50V had reduced intersubunit contacts, consistent with the increased K_d for dimer dissociation. Relative to PR, PR_I50V had fewer interactions of Val50 with inhibitors, in agreement with the dramatically increased K_i. 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.