Highly Drug-Resistant HIV-1 Protease Mutant PRS17 Shows Enhanced Binding to Substrate Analogues

We report the structural analysis of highly drug-resistant human immunodeficiency virus protease (PR) variant PR^S17, rationally selected by machine learning, in complex with substrate analogues. Crystal structures were solved of inhibitor-free inactive PR^S17-D25N, wild-type PR/CA-p2 complex, and PR^S17 in complex with substrate analogues, CA-p2 and p2-NC. Peptide analogues p2-NC and CA-p2 exhibit inhibition constants of 514 and 22 nM, respectively, for PR^S17 or approximately 3-fold better than for PR. CA-p2 is a better inhibitor of PR^S17 than are clinical inhibitors (K _i = 50-8390 nM) except for amprenavir (K _i = 11 nM). G48V resistance mutation induces curled flap tips in PR^S17-D25N structure. The inner P2-P2' residues of substrate analogues in PR^S17 complexes maintain similar conformations to those of wild-type complex, while significant conformational changes are observed in the peripheral residues P3, P4' of CA-p2 and P3, P4, and P3' of p2-NC. The loss of β-branched side chain by V82S mutation initiates a shift in 80's loop and reshapes the S3/S3' subsite, which enhances substrate binding with new hydrogen bonds and van der Waals interactions that are absent in the wild-type structures. The steric hindrance caused by G48V mutation in the flap of PR^S17 contributes to altered binding interactions of P3 Arg, P4' norleucine of CA-p2, and P4 and P3' of p2-NC with the addition of new hydrogen bonds and van der Waals contacts. The enhanced interaction of PR^S17 with substrate analogues agrees with their relative inhibition, suggesting that this mutant improves substrate binding while decreasing affinity for clinical inhibitors.