HIV-1 (human immunodeficiency virus type 1) protease (PR) and its mutants are important antiviral drug targets. The PR flap region is critical for binding substrates or inhibitors and catalytic activity. Hence, mutations of flap residues frequently contribute to reduced susceptibility to PR inhibitors in drug-resistant HIV. Structural and kinetic analyses were used to investigate the role of flap residues Gly48, Ile50, and Ile54 in the development of drug resistance. The crystal structures of flap mutants PRI50V(PR with I50V mutation), PRI54V(PR with I54V mutation), and PRI54M(PR with I54M mutation) complexed with saquinavir (SQV) as well as PRG48V(PR with G48V mutation), PRI54V, and PRI54Mcomplexed with darunavir (DRV) were determined at resolutions of 1.05-1.40 Å. The PR mutants showed changes in flap conformation, interactions with adjacent residues, inhibitor binding, and the conformation of the 80s loop relative to the wild-type PR. The PR contacts with DRV were closer in PRG48V-DRV than in the wild-type PR-DRV, whereas they were longer in PRI54M-DRV. The relative inhibition of PRI54Vand that of PRI54Mwere similar for SQV and DRV. PRG48Vwas about twofold less susceptible to SQV than to DRV, whereas the opposite was observed for PRI50V. The observed inhibition was in agreement with the association of G48V and I50V with clinical resistance to SQV and DRV, respectively. This analysis of structural and kinetic effects of the mutants will assist in the development of more effective inhibitors for drug-resistant HIV. © 2008 Elsevier Ltd. All rights reserved.
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