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Protein disulfide isomerase (PDI) is a very efficient catalyst of folding of many disulfide-bonded proteins. A great deal is known about the catalytic functions of PDI, while little is known about its substrate binding. We recently demonstrated by cross-linking that PDI binds peptides and misfolded proteins, with high affinity but broad specificity. To characterize the substrate-binding site of PDI, we investigated the interactions of various recombinant fragments of human PDI, expressed in Escherichia coli, with different radiolabelled model peptides. We observed that the b' domain of human PDI is essential and sufficient for the binding of small peptides. In the case of larger peptides, specifically a 28 amino acid fragment derived from bovine pancreatic trypsin inhibitor, or misfolded proteins, the b' domain is essential but not sufficient for efficient binding, indicating that contributions from additional domains are required. Hence we propose that the different domains of PDI all contribute to the binding site, with the b' domain forming the essential core.

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The formation of disulphide bonds in mammalian secretory and cell-surface proteins occurs in the lumen of the endoplasmic reticulum and is believed to be catalysed by the enzyme protein disulphide-isomerase (PDI). The evidence for this physiological role for PDI is circumstantial and relates to the cell and tissue distribution of the enzyme, its developmental behaviour and its catalytic properties in vitro. A clear requirement for PDI in the correct folding or assembly of disulphide-bonded proteins during biosynthesis has not been demonstrated. We have prepared dog pancreas microsomes which are deficient in soluble lumenal proteins, including PDI, but which are still able to translocate and process proteins synthesized in vitro. Using the formation of intramolecular disulphide bonds during the in vitro synthesis of gamma-gliadin, a wheat storage protein, as a model, we have demonstrated that these microsomes are defective in co-translational formation of disulphide bonds. Reconstitution of these microsomes with purified PDI reverses this defect.

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 fulllength 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.Native disulfide bond formation in the endoplasmic reticulum is a complex process that is rate-limiting in the biogenesis of many outer membrane and secreted proteins. Native disulfide bond formation can occur via multiple parallel pathways, and there is evidence that a large number of different gene families and redox carriers may play a role in the supply of redox equivalents for protein disulfide bond formation. What is clear is that the rate-limiting step for native disulfide bond formation in proteins that contain multiple disulfides is latestage isomerization reactions, where disulfide bond formation is linked to conformational changes in protein substrates with substantial regular secondary structure. These steps are thought to be catalyzed only by proteins belonging to the protein disulfide isomerase (PDI) family. PDI 1 was the first catalyst of protein folding identified over 40 years ago (1), but despite probably being the most widely studied protein folding catalyst, significant details of the mechanisms of action of this critical enzyme are still unclear. In all eukaryotes, there exists a species-dependent PDI family of enzymes; for example, in humans (2), ERp72, ERp57, P5, PDIp, PDIr, ERp44 (3), ERp28/29 (4), ERdj5 (5), and ERp18 (6) have been reported to date. Functional characterization and differentiation between these family members is far from complete. PDI is a multifunctional, multidomain enzyme. The domain structure of PDI has been determined by theoretical (7) and experimental (8 -11) procedures and comprises two catalytic domains, a and a, separated by two homologous non-catalytic domains, b and b, plus a C-terminal region designated as c. In addition, it has bee...