Alternative conformations of the x region of human protein disulphide-isomerase modulate exposure of the substrate binding b' domain

Nguyen, V.D. and Wallis, K. and Howard, M.J. and Haapalainen, A.M. and Salo, K.E. and Saaranen, M.J. and Sidhu, A. and Wierenga, R.K. and Freedman, R.B. and Ruddock, L.W. and Williamson, R.A. (2008) Alternative conformations of the x region of human protein disulphide-isomerase modulate exposure of the substrate binding b' domain. Journal of Molecular Biology, 383 (5). pp. 1144-1155. ISSN 0022-2836 . (The full text of this publication is not available from this repository)

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Official URL
http://dx.doi.org/10.1016/j.jmb.2008.08.085

Abstract

Protein disulphide isomerase (PDI) is a key multi-domain protein folding catalyst in the endoplasmic reticulum. The b' domain of PDI is essential for the non-covalent binding of incompletely folded protein substrates. Earlier, we defined the substrate binding site in the b' domain of human PDI by modelling and mutagenesis studies. Here, we show by fluorescence and NMR that recombinant human PDI b'x (comprising the b' domain and the subsequent x linker region) can assume at least two different conformations in solution. We have screened mutants in the b'x region to identify mutations that favour one of these conformers in recombinant b'x, and isolated and characterised examples of both types. We have crystallised one mutant of b'x (I272A mutation) in which one conformer is stabilized, and determined its crystal structure to a resolution of 2.2 A. This structure shows that the b' domain has the typical thioredoxin fold and that the x region can interact with the b' domain by "capping" a hydrophobic site on the b' domain. This site is most likely the substrate binding site and hence such capping will inhibit substrate binding. All of the mutations we previously reported to inhibit substrate binding shift the equilibrium towards the capped conformer. Hence, these mutations act by altering the natural equilibrium and decreasing the accessibility of the substrate binding site. Furthermore, we have confirmed that the corresponding structural transition occurs in the wild type full-length PDI. A cross-comparison of our data with that for other PDI-family members, Pdi1p and ERp44, suggests that the x region of PDI can adopt alternative conformations during the functional cycle of PDI action and that these are linked to the ability of PDI to interact with folding substrates.

Item Type: Article
Additional information: 1089-8638 (Electronic) Journal Article Research Support, Non-U.S. Gov't
Uncontrolled keywords: Amino Acid Sequence Crystallography, X-Ray Humans Magnetic Resonance Spectroscopy Models, Molecular Molecular Sequence Data Mutant Proteins/chemistry Mutation/genetics Protein Disulfide-Isomerases/*chemistry Protein Structure, Secondary Protein Structure, Tertiary Recombinant Proteins/chemistry Spectrometry, Fluorescence Substrate Specificity Tryptophan
Subjects: Q Science
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences > Protein Science Group
Depositing User: Sue Davies
Date Deposited: 13 May 2009 13:23
Last Modified: 13 May 2009 13:23
Resource URI: http://kar.kent.ac.uk/id/eprint/18444 (The current URI for this page, for reference purposes)
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