Peroxiredoxin-6 (PRDX6) is an unusual member of the peroxiredoxin family of antioxidant enzymes that has only one evolutionarily conserved cysteine. It reduces oxidized lipids and reactive oxygen species (ROS) by oxidation of the active-site cysteine (Cys47) to a sulfenic acid, but the mechanism for conversion back to a thiol is not completely understood. Moreover, it has phospholipase A2 (PLA2) activity in addition to its peroxidase activity. Interestingly, some biochemical data are inconsistent with a known high-resolution crystal structure of the catalytic intermediate of the protein, and biophysical data indicate that the protein undergoes conformational changes that affect enzyme activity. In order to further elucidate the solution structure of this important enzyme, we used chemical cross-linking coupled with high-resolution MS (CX–MS), with an emphasis on zero-length cross-links. Distance constraints from high confidence cross-links were used in homology modelling experiments to determine a solution structure of the reduced form of the protein. This structure was further evaluated using chemical cross-links produced by several homo-bifunctional amine-reactive cross-linking reagents, which helped to confirm the solution structure. The results show that several regions of the reduced version of human PRDX6 are in a substantially different conformation from that shown for the crystal structure of the peroxidase catalytic intermediate. The differences between these two structures are likely to reflect catalysis-related conformational changes. These studies also demonstrate that CX–MS using zero-length cross-linking is a powerful strategy for probing protein conformational changes that is complementary to alternative methods such as crystallographic, NMR and biophysical studies.
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Research Article|
May 05 2015
Solution structure of the reduced form of human peroxiredoxin-6 elucidated using zero-length chemical cross-linking and homology modelling
Roland F. Rivera-Santiago;
Roland F. Rivera-Santiago
*The Center for Systems and Computational Biology, The Wistar Institute and Molecular and Cellular Oncogenesis, Philadelphia, PA 19104, U.S.A.
†Biochemistry and Molecular Biophysics Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.
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Sandra L. Harper;
Sandra L. Harper
*The Center for Systems and Computational Biology, The Wistar Institute and Molecular and Cellular Oncogenesis, Philadelphia, PA 19104, U.S.A.
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Suiping Zhou;
Suiping Zhou
#x2021;Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104, U.S.A.
§Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.
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Sira Sriswasdi;
Sira Sriswasdi
*The Center for Systems and Computational Biology, The Wistar Institute and Molecular and Cellular Oncogenesis, Philadelphia, PA 19104, U.S.A.
║Department of Biological Sciences, Graduate School of Sciences, University of Tokyo, Tokyo 113-0032, Japan
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Sheldon I. Feinstein;
Sheldon I. Feinstein
#x2021;Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104, U.S.A.
§Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.
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Aron B. Fisher;
Aron B. Fisher
#x2021;Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104, U.S.A.
§Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.
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David W. Speicher
David W. Speicher
1
*The Center for Systems and Computational Biology, The Wistar Institute and Molecular and Cellular Oncogenesis, Philadelphia, PA 19104, U.S.A.
†Biochemistry and Molecular Biophysics Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.
1To whom correspondence should be addressed (email speicher@wistar.org).
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Publisher: Portland Press Ltd
Received:
December 03 2014
Revision Received:
February 17 2015
Accepted:
March 06 2015
Accepted Manuscript online:
March 06 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2015 Biochemical Society
2015
Biochem J (2015) 468 (1): 87–98.
Article history
Received:
December 03 2014
Revision Received:
February 17 2015
Accepted:
March 06 2015
Accepted Manuscript online:
March 06 2015
Citation
Roland F. Rivera-Santiago, Sandra L. Harper, Suiping Zhou, Sira Sriswasdi, Sheldon I. Feinstein, Aron B. Fisher, David W. Speicher; Solution structure of the reduced form of human peroxiredoxin-6 elucidated using zero-length chemical cross-linking and homology modelling. Biochem J 15 May 2015; 468 (1): 87–98. doi: https://doi.org/10.1042/BJ20141463
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