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Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase.

By Annamari Pirneskoski, Peter Klappa, Mario Lobell, Richard A. Williamson, Lee J. Byrne, Heli I. Alanen, Kirsi E. H. Salo, Kari I. Kivirikkos, Robert B. Freedman and Lloyd W. Ruddock

Abstract

Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b' domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b' domain and show the existence of an intradomain linker between the b' and a' domains. We have expressed the recombinant b' domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b' domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect

Topics: Q
Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3996 USA
Year: 2004
OAI identifier: oai:kar.kent.ac.uk:47
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