3rd International AIDS Society Conference on HIV Pathogenesis and Treatment Rio de Janeiro - July 24 - 27, 2005

UNDERSTANDING THE ROLE OF INVARIANT THR80 IN HIV-1 PROTEASE

IAS Conf HIV Pathog Treat 2005 Jul 24-27;3rd: Abstract No. WePp0101

Foulkes J.¹, O'Brien S.¹, Prabu-Jeyabalan M.¹, Osterhout J.J.², Schiffer C.A.^1
1 University of Massachusetts Medical School, Worcester, MA, United States of America, ² University of Arizona, Tucson, AZ, United States of America

INTRODUCTION: All known protease inhibitors target the active site of protease. However, protease tolerates mutations within the active site that impair its ability to bind inhibitors without affecting its activity leading to drug resistance. Amino acids within the protease sequence that are invariant in the patient population thus far maybe crucial to the structure or function of protease. Further examination of these invariant amino acids may reveal new ways to target protease for development of novel inhibitors.

METHODS: Four variants of HIV-1 protease were created through site-directed mutagenesis: T80V, T80S, T80N, and T80A. They were examined for structural changes by circular dichroism spectroscopy and protein crystallography, and for functional changes by the ability to cleave a protease substrate, and to bind the protease inhibitor saquinavir.

RESULTS: The following results indicate that most mutations at threonine 80 substantially impact protease function without significantly altering structure. CD spectroscopy showed T80S, T80N, and T80V were structurally similar to wild-type. T80A was less structured than wild-type. Protein crystallography indicates T80S and T80N had similar overall structure to wild-type bound to saquinavir, though both variants had increased van der Waals contacts between the protease and saquinavir. Each variant's activity was measured as the kcat/Km versus a substrate of protease. T80S was 1.2 times more active than wild-type, while T80V was an order of magnitude less active. Activity levels for T80A and T80N were not measurable. T80S and T80V bound saquinavir as wild-type, while T80N binding was decreased by three orders of magnitude. T80A did not bind saquinavir.

CONCLUSIONS: Threonine is the optimal amino acid at position 80. While protease can tolerate serine, mutation to valine, asparagine, or alanine significantly alters the function. This study demonstrates the functional relevance of this invariant residue, and the importance of studying other invariant residues in protease.

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Basic | WePp0101 | Jennifer Foulkes
Mechanisms Of Drug Resistance

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