MOLECULAR-BASIS OF HIV-1 PROTEASE DRUG-RESISTANCE - STRUCTURAL-ANALYSIS OF MUTANT PROTEASES COMPLEXED WITH CYCLIC UREA INHIBITORS

In cell cultures, the key residues associated with HIV-1 resistance to cyclic urea-based HIV-1 protease (PR) inhibitors are Val82 and Ile84 of HIV-1 PR. To gain an understanding of how these two residues modula te inhibitor binding, we have measured the K-i values of three recombi nant mutant proteases, I84V, V82F, and V82F/I84V, for DMP323 and DMP45 0, and determined the three-dimensional structures of their complexes to 2.1-1.9 Angstrom resolution with R factors of 18.7-19.6%. The K-i v alues of these mutants increased by 25-, 0.5-, and 1000-fold compared to the wild-type values of 0.8 and 0.4 nM for DMP323 and DMP450, respe ctively. The wild-type and mutant complexes overall are very similar ( rms deviations of 0.2-0.3 Angstrom) except for differences in the patt erns of their van der Waals (vdw) interactions, which appear to modula te the K-i values of the mutants. The loss of the CD1 atom of Ile84, i n the I84V mutant complexes, creates a hole in the S1 subsite, reducin g the number of vdw contacts and increasing the K-i values. The V82F m utant binds DMP323 more tightly than wild type because the side chain of Phe82 forms additional vdw and edge-to-face interactions with the P 1 group of DMP323. The K-i values of the single mutants are not additi ve because the side chain of Phe82 rotates out of the S1 subsite in th e double mutant (the chi(1) angles of Phe82 and -182 in the V82F and V 82F/I84V mutants differ by 90 and 185 degrees, respectively), further reducing the vdw interactions. Finally, compensatory shifts in the I84 V and V82F/I84V complexes pick up a small number of new contacts, but too few to offset the initial loss of interactions caused by the mutat ions. Therefore, our data suggest that variants persist in the presenc e of DMP323 and DMP450 because of a decrease in vdw interactions betwe en the mutant proteases and inhibitors.