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...

The protein disulfide isomerase (PDI) family of folding catalysts are constructed from combinations of redoxactive and redox-inactive domains, all of which are probably based on the thioredoxin fold. To understand the function of each domain in the variety of catalytic reactions that each family member can perform (to differing extents), the domain boundaries of each family member must be known. By using a technique based on sequence alignments and the known structure of the a and b domains of human PDI, we generated a large number of domain constructs for all six redox-active human PDIs: PDI, PDIp, ERp72, ERp57, P5, and PDIr. The ability to generate significant amounts of soluble protein in E. coli from most of these domain constructs strongly indicates that the domain boundaries are correct. The implications for these domain boundaries on the tertiary structure of the human PDIs are discussed.

Protein disulfide isomerase (PDI) is a modular polypeptide consisting of four domains, a, b, b, and a, plus an acidic C-terminal extension, c. PDI carries out multiple functions, acting as the ␤ subunit in the animal prolyl 4-hydroxylases and in the microsomal triglyceride transfer protein and independently acting as a protein folding catalyst. We report here that the minimum sequence requirement for the assembly of an active prolyl 4-hydroxylase ␣ 2 ␤ 2 tetramer in insect cell coexpression experiments is fulfilled by the PDI domain construct ba but that the sequential addition of the b and a domains greatly increases the level of enzyme activity obtained. In the assembly of active prolyl 4-hydroxylase tetramers, the a and b domains of PDI, but not b and a, can in part be substituted by the corresponding domains of ERp57, a PDI isoform that functions naturally in association with the lectins calnexin and calreticulin. The a domain of PDI could not be substituted by the PDI a domain, suggesting that both b and a domains contain regions critical for prolyl 4-hydroxylase assembly. All PDI domain constructs and PDI/ERp57 hybrids that contain the b domain can bind the 14-amino acid peptide ⌬-somatostatin, as measured by cross-linking; however, binding of the misfolded protein "scrambled" RNase required the addition of domains ab or a of PDI. The human prolyl 4-hydroxylase ␣ subunit has at least two isoforms, ␣(I) and ␣(II), which form with the PDI polypeptide the (␣(I)) 2 ␤ 2 and (␣(II)) 2 ␤ 2 tetramers. We report here that all the PDI domain constructs and PDI/ERp57 hybrid polypeptides tested were more effectively associated with the ␣(II) subunit than the ␣(I) subunit. Protein disulfide isomerase (PDI)1 (EC 5.3.4.1), a major protein within the lumen of the eukaryotic endoplasmic reticulum, is a catalyst of disulfide bond formation and rearrangement in protein folding (for reviews, see Refs. 1 and 2). PDI is a modular protein consisting of four domains, a, b, bЈ, and aЈ, plus an acidic C-terminal extension, c (3, 4). The a and aЈ domains show sequence similarity to thioredoxin, contain the catalytic site motif CGHC (1), and have the thioredoxin fold (3, 5). The b and bЈ domains show no amino acid sequence similarity to thioredoxin and have no catalytic site sequence, but recent NMR studies have indicated that the b domain (and by homology the bЈ domain) also has the thioredoxin fold (2, 6).PDI is a multifunctional polypeptide. In addition to its role in protein folding within the endoplasmic reticulum (12-20), PDI serves as the ␤ subunit in the animal prolyl 4-hydroxylase ␣ 2 ␤ 2 tetramers and ␣␤ dimers (7-9) and in the microsomal triglyceride transfer protein ␣␤ dimer (10, 11). Prolyl 4-hydroxylase plays a central role in the synthesis of all collagens (8, 9, 21), whereas the microsomal triglyceride transfer protein is essential for the assembly of apoB-containing lipoproteins (10, 11). The main function of PDI in both of these proteins appears to be to keep their highly insoluble ␣ subunits in a catalytically acti...

Protein disulfide isomerase (PDI) is a multifunctional polypeptide that acts as a subunit in the animal prolyl 4-hydroxylases and the microsomal triglyceride transfer protein, and as a chaperone that binds various peptides and assists their folding. We report here that deletion of PDI sequences corresponding to the entire C-terminal domain c, previously thought to be critical for chaperone activity, had no inhibitory effect on the assembly of recombinant prolyl 4-hydroxylase in insect cells or on the in vitro chaperone activity or disulfide isomerase activity of purified PDI. However, partially overlapping critical regions for all these functions were identified at the C-terminal end of the preceding thioredoxinlike domain aЈ. Point mutations introduced into this region identified several residues as critical for prolyl 4-hydroxylase assembly. Circular dichroism spectra of three mutants suggested that two of these mutations may have caused only local alterations, whereas one of them may have led to more extensive structural changes. The critical region identified here corresponds to the C-terminal α helix of domain aЈ, but this is not the only critical region for any of these functions.