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Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease

Alvizo, Oscar and Mittal, Seema and Mayo, Stephen L. and Schiffer, Celia A. (2012) Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease. Protein Science, 21 (7). pp. 1029-1041. ISSN 0961-8368. http://resolver.caltech.edu/CaltechAUTHORS:20120712-085217104

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Abstract

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT–RH over p2-NC and CA-p2. In kinetic assays, RT–RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT–RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1002/pro.2086 DOIUNSPECIFIED
http://onlinelibrary.wiley.com/doi/10.1002/pro.2086/abstractPublisherUNSPECIFIED
Additional Information:© 2012 The Protein Society. Published by Wiley-Blackwell. Received 18 January 2012; Revised 23 March 2012; Accepted 10 April 2012. Article first published online: 5 Jun. 2012. Grant sponsor: National Institutes of Health; Grant numbers: R01 GM064347P01 GM66524; Grant sponsor: National Institutes of Health, National Center for Research Resources; Grant number: RR007707; Grant sponsors: Office of Biological and Environmental Research; Office of Basic Energy Sciences of the US Department of Energy; National Center for Research Resources of the National Institutes of Health. The authors thank Dr. Vukica Srajer at BioCARS, sector 14 Advanced Photon Source at Argonne National Laboratory for help with data collection, Dr. William E. Royer and Dr. Madhavi N. Nalam for assistance with initial refinement, and Dr. Nese Kurt Yilmaz and Ms. Marie Ary for editorial assistance. They thank Annie Heroux, beam line scientist at the Macromolecular Crystallography Research Resource (PXRR) of the Brookhaven National Laboratory, for collecting some of the data at beamline X25 of the National Synchrotron Light Source through the mail-in crystal program.
Funders:
Funding AgencyGrant Number
NIHR01 GM064347P01
NIHGM66524
NIH National Center for Research ResourcesRR007707
Department of Energy (DOE) Office of Basic Energy ScienceUNSPECIFIED
NIH National Center for Research ResourcesUNSPECIFIED
Subject Keywords:HIV-1 protease; positive design; substrate specificity; X-ray crystallography; isothermal titration calorimetry; substrate envelope
Record Number:CaltechAUTHORS:20120712-085217104
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20120712-085217104
Official Citation:Alvizo, O., Mittal, S., Mayo, S. L. and Schiffer, C. A. (2012), Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease. Protein Science, 21: 1029–1041. doi: 10.1002/pro.2086
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:32372
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:12 Jul 2012 21:30
Last Modified:23 Aug 2016 10:14

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