title: Dimerization of HIV-1 protease occurs through two steps relating to the mechanism of protease dimerization inhibition by darunavir.
abstract: Dimerization of HIV-1 protease (PR) subunits is an essential process for PR's acquisition of proteolytic activity, which plays a critical role in the maturation of HIV-1. Recombinant wild-type PR (PR(WT)) proved to dimerize, as examined with electrospray ionization mass spectrometry; however, two active site interface PR mutants (PR(T26A) and PR(R87K)) remained monomeric. On the other hand, two termini interface PR mutants (PR(1-C95A) and PR(97/99)) took both monomeric and dimeric forms. Differential scanning fluorimetry indicated that PR(1-C95A) and PR(97/99) dimers were substantially less stable than PR(WT) dimers. These data indicate that intermolecular interactions of two monomers occur first at the active site interface, generating unstable or transient dimers, and interactions at the termini interface subsequently occur, generating stable dimers. Darunavir (DRV), an HIV-1 protease inhibitor, inhibits not only proteolytic activity but also PR dimerization. DRV bound to protease monomers in a one-to-one molar ratio, inhibiting the first step of PR dimerization, whereas conventional protease inhibitors (such as saquinavir) that inhibit enzymatic activity but not dimerization failed to bind to monomers. DRV also bound to mutant PRs containing the transframe region-added PR (TFR-PR(D25N) and TFR-PR(D25N-7AA)), whereas saquinavir did not bind to TFR-PR(D25N) or TFR-PR(D25N-7AA). Notably, DRV failed to bind to mutant PR containing four amino acid substitutions (V32I, L33F, I54M, and I84V) that confer resistance to DRV on HIV-1. To our knowledge, the present report represents the first demonstration of the two-step PR dimerization dynamics and the mechanism of dimerization inhibition by DRV, which should help design further, more potent novel PIs.
authors.affiliation: Departments of Hematology, Rheumatology, and Infectious Disease, Kumamoto University Graduate School of Medicine, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan;Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Hironori
authors.initials: H
authors.lastname: Hayashi
authors.affiliation: Department of Biochemistry, Kumamoto University Graduate School of Pharmaceutical Sciences, 5-1 Ooehonmachi, Chuo-ku, Kumamoto 862-0973, Japan;
authors.forename: Nobutoki
authors.initials: N
authors.lastname: Takamune
authors.affiliation: Bruker Daltonics K.K., 3-9 Moriya, Kanagawa-ku, Yokohama 221-0022, Japan;
authors.forename: Takashi
authors.initials: T
authors.lastname: Nirasawa
authors.affiliation: Departments of Hematology, Rheumatology, and Infectious Disease, Kumamoto University Graduate School of Medicine, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan;Department of Medical Technology, Kumamoto Health Science University, 325 Izumi-cho, Kita-ku, Kumamoto 861-5598, Japan; and.
authors.forename: Manabu
authors.initials: M
authors.lastname: Aoki
authors.affiliation: Bruker Daltonics K.K., 3-9 Moriya, Kanagawa-ku, Yokohama 221-0022, Japan;
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Morishita
authors.affiliation: Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Debananda
authors.initials: D
authors.lastname: Das
authors.affiliation: Departments of Hematology, Rheumatology, and Infectious Disease, Kumamoto University Graduate School of Medicine, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan;
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Koh
authors.affiliation: Departments of Chemistry and Medicinal Chemistry, Purdue University, West Lafayette, IN 47907.
authors.forename: Arun K
authors.initials: AK
authors.lastname: Ghosh
authors.affiliation: Department of Biochemistry, Kumamoto University Graduate School of Pharmaceutical Sciences, 5-1 Ooehonmachi, Chuo-ku, Kumamoto 862-0973, Japan;
authors.forename: Shogo
authors.initials: S
authors.lastname: Misumi
authors.affiliation: Departments of Hematology, Rheumatology, and Infectious Disease, Kumamoto University Graduate School of Medicine, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan;Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; hmitsuya@helix.nih.gov.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mitsuya
mesh_terms: D020134:Catalytic Domain; D000069454:Darunavir; D019281:Dimerization; D016333:HIV Protease / Q000737:chemistry*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D008958:Models, Molecular; D021241:Spectrometry, Mass, Electrospray Ionization; D013449:Sulfonamides / Q000494:pharmacology*
chemical_list: D017320:HIV Protease Inhibitors; D013449:Sulfonamides; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1; D000069454:Darunavir
keywords: AIDS; protease precursor; thermal stability; two-step dimerization dynamics
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