Structural implications of drug-resistant mutants of HIV-1 protease: High-resolution crystal structures of the mutant protease/substrate analogue complexes

Emergence of drug-resistant mutants of HIV-1 protease is an ongoing problem in the fight against AIDS. The mechanisms governing resistance are both co mplex and varied. We have determined crystal structures of HIV-1 protease m utants, D30N, K45I, N88D, and L90M complexed with peptide inhibitor analogu es of CA-p2 and p2-NC cleavage sites in the Gag-pol precursor in order to s tudy the structural mechanisms underlying resistance. The structures were d etermined at 1.55-1.9-Angstrom resolution and compared with the wild-type s tructure. The conformational disorder seen for most of the hydrophobic side -chains around the inhibitor binding site indicates flexibility of binding. Eight water molecules are conserved in all 9 structures; their location su ggests that they are important for catalysis as well as structural stabilit y. Structural differences among the mutants were analyzed in relation to th e observed changes in protease activity and stability. Mutant L90M shows st eric contacts with the catalytic Asp25 that could destabilize the catalytic loop at the dimer interface, leading to its observed decreased dimer stabi lity and activity. Mutant K45I reduces the mobility of the flap and the inh ibitor and contributes to an enhancement in structural stability and activi ty. The side-chain variations at residue 30 relative to wild-type are the l argest in D30N and the changes are consistent with the altered activity obs erved with peptide substrates, Polar interactions in D30N are maintained, i n agreement with the observed urea sensitivity. The side-chains of D30N and N88D are linked through a water molecule suggesting correlated changes at the two sites, as seen with clinical inhibitors. Structural changes seen in N88D are small; however, water molecules that mediate interactions between Asn88 and Thr74/Thr31/Asp30 in other complexes are missing in N88D, Protei ns 2001;43:455-464. (C) 2001 Wiley-Liss, Inc.