Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 396, Issue 2, Pages 361-374Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2009.11.049
Keywords
protein disulfide isomerase; NMR; SAXS; molecular chaperone; domain rearrangement
Categories
Funding
- Japan Society for the Promotion of Science
- Ministry of Education, Culture, Sports, Science and Technology of Japan [15032249, 17028047, 15076210, 18870023, 20050030, 20059030, 20107004, 21370050, 21870052]
- Institute for Molecular Science from National Institute of Natural Sciences
- Grants-in-Aid for Scientific Research [17028047, 21370050, 18870023, 20059030, 15032249, 20107004, 21870052] Funding Source: KAKEN
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Protein disulfide isomerase (PDI) is a major protein in the endoplasmic reticulum, operating as an essential folding catalyst and molecular chaperone for disulfide-containing proteins by catalyzing the formation, rearrangement, and breakage of their disulfide bridges. This enzyme has a modular structure with four thioredoxin-like domains, a, b, b', and a', along with a C-terminal extension. The homologous a and a' domains contain one cysteine pair in their active site directly involved in thiol-disulfide exchange reactions, while the b' domain putatively provides a primary binding site for unstructured regions of the substrate polypeptides. Here, we report a redox-dependent intramolecular rearrangement of the b' and a' domains of PDI from Humicola insolens, a thermophilic fungus, elucidated by combined use of nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS) methods. Our NMR data showed that the substrates bound to a hydrophobic surface spanning these two domains, which became more exposed to the solvent upon oxidation of the active site of the a' domain. The hydrogen-deuterium exchange and relaxation data indicated that the redox state of the a' domain influences the dynamic properties of the W domain. Moreover, the SAXS profiles revealed that oxidation of the a' active site causes segregation of the two domains. On the basis of these data, we propose a mechanistic model of PDI action; the a' domain transfers its own disulfide bond into the unfolded protein accommodated on the hydrophobic surface of the substrate-binding region, which consequently changes into a closed form releasing the oxidized substrate. (C) 2009 Elsevier Ltd. All rights reserved.
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