HIV-1 protease is a key target in treating
HIV infection and
AIDS, with 10 inhibitors used clinically. Here we used an unusual hexapeptide substrate, containing two macrocyclic tripeptides constrained to mimic a beta strand conformation, linked by a scissile
peptide bond, to probe the structural mechanism of proteolysis. The substrate has been cocrystallized with catalytically active synthetic
HIV-1 protease and an inactive isosteric (D25N) mutant, and three-dimensional structures were determined (1.60 A). The structure of the inactive HIVPR(D25N)/substrate complex shows an intact substrate molecule in a single orientation that perfectly mimics the binding of conventional
peptide ligands of HIVPR. The structure of the active HIVPR/product complex shows two monocyclic hydrolysis products trapped in the active site, revealing two molecules of the N-terminal monocyclic product bound adjacent to one another, one molecule occupying the nonprime site, as expected, and the other monocycle binding in the prime site in the reverse orientation. The results suggest that both hydrolysis products are released from the active site upon cleavage and then rebind to the
enzyme. These structures reveal that N-terminal binding of
ligands is preferred, that the C-terminal site is more flexible, and that HIVPR can recognize substrate shape rather than just sequence alone. The product complex reveals three
carboxylic acids in an almost planar orientation, indicating an unusual hexagonal homodromic complex between three
carboxylic acids. The data presented herein regarding orientation of catalytic aspartates support the cleavage mechanism proposed by Northrop. The results imply strategies for design of inhibitors targeting the N-terminal side of the cleavage site or taking advantage of the flexibility in the
protease domain that accommodates substrate/inhibitor segments C-terminal to the cleavage site.