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 modulate inhibitor binding, we have measured the K i values of three recombinant mutant proteases, I84V, V82F, and V82F/I84V, for DMP323 and DMP450, and determined the three-dimensional structures of their complexes to 2.1−1.9 Å resolution with R factors of 18.7−19.6%. The K i values 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, respectively. The wild-type and mutant complexes overall are very similar (rms deviations of 0.2−0.3 Å) except for differences in the patterns of their van der Waals (vdw) interactions, which appear to modulate the K i values of the mutants. The loss of the CD1 atom of Ile84, in the I84V mutant complexes, creates a hole in the S1 subsite, reducing the number of vdw contacts and increasing the K i values. The V82F mutant binds DMP323 more tightly than wild type because the side chain of Phe82 forms additional vdw and edge-to-face interactions with the P1 group of DMP323. The K i values of the single mutants are not additive because the side chain of Phe82 rotates out of the S1 subsite in the double mutant (the χ1 angles of Phe82 and -182 in the V82F and V82F/I84V mutants differ by 90 and 185°, respectively), further reducing the vdw interactions. Finally, compensatory shifts in the I84V 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 mutations. Therefore, our data suggest that variants persist in the presence of DMP323 and DMP450 because of a decrease in vdw interactions between the mutant proteases and inhibitors.