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 (PRWT) proved to dimerize, as examined with electrospray ionization mass spectrometry; however, two active site interface PR mutants (PRT26A and PRR87K) remained monomeric. On the other hand, two termini interface PR mutants (PR 1-C95A and PR97/99) took both monomeric and dimeric forms. Differential scanning fluorimetry indicated that PR1-C95A and PR97/99 dimers were substantially less stable than PRWT 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-PRD25N and TFR-PRD25N-7AA), whereas saquinavir did not bind to TFR-PRD25N or TFR-PRD25N-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.