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Crystal structures of highly constrained substrate and hydrolysis products bound to HIV-1 Protease. Implications for the catalytic mechanism

Tyndall, Joel D. A. , Pattenden, Leonard Keith, Reid, Robert, Hu, Shu-Hong , Alewood, Dianne , Alewood, Paul , Walsh, Terence P., Fairlie, David P. , & Martin, Jennifer (2008) Crystal structures of highly constrained substrate and hydrolysis products bound to HIV-1 Protease. Implications for the catalytic mechanism. Biochemistry, 47(12), pp. 3736-3744.

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Abstract

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 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 Å). 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.

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9 citations in Web of Science®

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ID Code: 18393
Item Type: Journal Article
Additional Information: This article is freely available from the American Chemical Society website 12 months after the publication date. See links to publisher website in this record.
Keywords: HIV-1, protease, structure, catalysis
DOI: 10.1021/bi7023157
ISSN: 0006-2960
Divisions: Past > QUT Faculties & Divisions > Faculty of Science and Technology
Current > Institutes > Institute of Health and Biomedical Innovation
Past > Schools > School of Life Sciences
Copyright Owner: Copyright 2008 American Chemical Society
Deposited On: 03 Mar 2009 15:11
Last Modified: 29 Feb 2012 23:48

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