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 K., Reid, Robert C., Hu, Shu-Hong, Alewood, Dianne, Alewood, Paul F., Walsh, Terry, Fairlie, David P. and Martin, Jennifer L. (2008) Crystal structures of highly constrained Substrate and Hydrolysis products bound to HIV-1 Protease. Implications for the Catalytic Mechanism. Biochemistry, 47 12: 3736-3744. doi:10.1021/bi7023157


Author Tyndall, Joel D. A.
Pattenden, Leonard K.
Reid, Robert C.
Hu, Shu-Hong
Alewood, Dianne
Alewood, Paul F.
Walsh, Terry
Fairlie, David P.
Martin, Jennifer L.
Title Crystal structures of highly constrained Substrate and Hydrolysis products bound to HIV-1 Protease. Implications for the Catalytic Mechanism
Journal name Biochemistry   Check publisher's open access policy
ISSN 0006-2960
Publication date 2008-01-17
Year available 2008
Sub-type Article (original research)
DOI 10.1021/bi7023157
Volume 47
Issue 12
Start page 3736
End page 3744
Total pages 9
Place of publication United States
Publisher American Chemical Soc
Collection year 2009
Language eng
Subject 270199 Biochemistry and Cell Biology not elsewhere classified

C1
970103 Expanding Knowledge in the Chemical Sciences
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 β 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.
Q-Index Code C1
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Created: Tue, 03 Feb 2009, 16:52:11 EST by Cody Mudgway on behalf of Institute for Molecular Bioscience