Abstract
Drug resistance of HIV-1 protease alters the balance in the molecular recognition events in favor of substrate processing versus inhibitor binding. To develop robust inhibitors targeting ensembles of drug-resistant variants, the code of this balance needs to be cracked. For this purpose, the principles governing the substrate recognition are required to be revealed. Previous crystallographic studies on the WT protease-substrate complexes showed that the substrates have a conserved consensus volume in the protease active site despite their low sequence homology. This consensus volume is termed as the substrate envelope. The substrate envelope was recently reevaluated by taking the substrate dynamics into account, and the dynamic substrate envelope was reported to better define the substrate specificity for HIV-1 protease. Drug resistance occurs mostly through mutations in the protease, occasionally accompanied by cleavage site mutations. In this study, three coevolved protease-substrate complexes ((AP2V)NC-p1(V82A), (LP1'F)p1-p6(D30N/N88D), and (SP3'N)p1-p6(D30N/N88D)) were investigated for structural and dynamic properties by molecular modeling and dynamics simulations. The results show the substrate envelope is preserved by these cleavage site mutations in the presence of drug-resistance mutations in the protease, if not enhanced. This study on the conformational and mutational ensembles of protease-substrate complexes validates the substrate envelope as the substrate recognition motif for HIV-1 protease. The substrate envelope hypothesis allows for the elucidation of possible drug resistance mutation patterns in the polyprotein cleavage sites.