title: 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.
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2050, South Africa.
authors.forename: Previn
authors.initials: P
authors.lastname: Naicker
authors.forename: Palesa
authors.initials: P
authors.lastname: Seele
authors.forename: Heini W
authors.initials: HW
authors.lastname: Dirr
authors.forename: Yasien
authors.initials: Y
authors.lastname: Sayed
mesh_terms: D000409:Alanine; D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics; D016333:HIV Protease / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D055503:Protein Multimerization / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1; D000409:Alanine