F99 is Critical for Dimerization and Activation of South African HIV-1 Subtype C Protease

Abstract

HIV-1 protease (PR) is an obligate homodimer which plays a pivotal role in the maturation and hence propagation of HIV. Although successful developments on PR active site inhibitors have been achieved, the major limiting factor has been the emergence of HIV drug-resistant strains. Disruption of the dimer interface serves as an alternative mechanism to inactivate the enzyme. The terminal residue, F99, was mutated to an alanine to investigate its contribution to dimer stability in the South African HIV-1 subtype C (C-SA) PR. The F99A PR and wild-type C-SA PR were overexpressed and purified. The activities of the PRs and their ability to bind an active site inhibitor, acetyl-pepstatin, were determined in vitro. The F99A PR showed no activity and the inability to bind to the inhibitor. Secondary and quaternary structure analysis were performed and revealed that the F99A PR is monomeric with reduced β-sheet content. The mutation of F99 to alanine disrupted the presumed 'lock-and-key' motif at the terminal dimer interface, in turn creating a cavity at the N- and C-terminal antiparallel β-sheet. These findings support the design of inhibitors targeting the C-terminus of the C-SA PR, centered on interactions with the bulky F99.

Amide hydrogen exchange in HIV–1 subtype B and C proteases – insights into reduced drug susceptibility and dimer stability

“…Therefore, these residues may contribute less to the stability of the N‐ and C–termini β–sheet than other C–terminal residues. Mutation of the ultimate amino acid in the HIV–1 protease sequence, phenylalanine, to an alanine was shown to reduce the β–sheet content of the protease and disrupt dimerization, which results in complete loss of activity . Contacts made by F99 with neighbouring residues are shown in Fig.…”