Three Binding Sites in Protein-disulfide Isomerase Cooperate in Collagen Prolyl 4-Hydroxylase Tetramer Assembly

Abstract

Protein-disulfide isomerase (PDI) is a modular polypeptide consisting of four domains, a, b, b , and a . It is a ubiquitous protein folding catalyst that in addition functions as the ␤-subunit in vertebrate collagen prolyl 4-hydroxylase (C-P4H) ␣ 2 ␤ 2 tetramers. We report here that point mutations in the primary peptide substrate binding site in the b domain of PDI did not inhibit C-P4H assembly. Based on sequence conservation, additional putative binding sites were identified in the a and a domains. Mutations in these sites significantly reduced C-P4H tetramer assembly, with the a domain mutations generally having the greater effect. When the a or a domain mutations were combined with the b domain mutation I272W tetramer assembly was further reduced, and more than 95% of the assembly was abolished when mutations in the three domains were combined. The data indicate that binding sites in three PDI domains, a, b , and a , contribute to efficient C-P4H tetramer assembly. The relative contributions of these sites were found to differ between Caenorhabditis elegans C-P4H ␣␤ dimer and human ␣ 2 ␤ 2 tetramer formation.Protein-disulfide isomerase (PDI 1 ; EC 5.3.4.1) is a ubiquitous catalyst of disulfide bond formation and rearrangement during protein folding in the endoplasmic reticulum (ER). In addition, it functions as a subunit in two enzyme complexes, the collagen prolyl 4-hydroxylases (C-P4Hs; EC 1.14.11.2) (1, 2) and microsomal triglyceride transfer protein (3). The C-P4Hs, which are crucial for the synthesis of extracellular collagen macromolecules (4), are tetrameric enzymes consisting of two catalytic ␣ subunits and two ␤ subunits identical to PDI (5). The role of PDI in this tetramer is to keep the highly insoluble ␣ subunits in solution and in a catalytically active, nonaggregated conformation (6, 7). In addition, PDI retains the tetramer within the ER, since the ␣ subunits lack the ER retrieval signal. The human C-P4Hs have at least three isoenzymes, ␣(I) 2 ␤ 2 , ␣(II) 2 ␤ 2 , and ␣(III) 2 ␤ 2 , in which the ␣ subunits differ but the ␤ subunit is always PDI (8 -11).The PDI polypeptide comprises four domains, a, b, bЈ, and aЈ, of which the first and last contain the -CGHC-catalytic site required for thiol-disulfide exchange activity (12) (see Fig. 1). The a and b domains have a thioredoxin fold (13,14), and the bЈ and aЈ domains are also thought on the basis of homology to have the same fold. In addition, there is a short linker region x between the bЈ and aЈ domains (15), and the C terminus of the polypeptide contains a highly acidic 29-amino acid extension c, which is not critical for any of the major functions of the protein except ER retrieval (16). The principle substrate binding site of PDI is located in the bЈ domain (17), which by itself is capable of binding short peptide substrates. We have recently identified a putative hydrophobic pocket in the bЈ domain by structural homology modeling and sequence alignments and shown by site-directed mutagenesis that this pocket contains several residues that...

ERp57 Does Not Require Interactions with Calnexin and Calreticulin to Promote Assembly of Class I Histocompatibility Molecules, and It Enhances Peptide Loading Independently of Its Redox Activity

“…Another example is clusterin, which, following in vitro translation in the presence of castanospermine, could still be detected in a noncovalent complex with ERp57 (42). By analogy to PDI, ERp57 may possess secondary substrate binding sites in the a and aЈ domains (38,39,45,46). Consistent with this, Ellgaard and co-workers (5) have demonstrated that the isolated a and aЈ domains possess disulfide isomerase activity that is capable of reactivating disulfide-scrambled ribonuclease A in vitro.…”

Annular Arrangement and Collaborative Actions of Four Domains of Protein-disulfide Isomerase

“…As the resolution in the models constructed using SAXS is limited, and the NMR structures of the domain a and b were used to replace the structures of aЈ and bЈ, the restored model presented here is reasonable and very probable but does not necessarily represent a unique solution to modeling the data. Many experimental data have shown that all four domains cooperate with each other to participate in binding of the protein target (22,43,44), although the bЈ domain contributes to the principal peptide-binding site (45). All four domains have been found to be required for PDI to perform efficiently as a chaperone (22,43,44) and to exhibit maximum catalytic activity (21).…”

“…This suggests that other parts of the PDI molecule are required for its full range of activities. A binding site in the a domain has been found recently to contribute to prolyl 4-hydroxylase tetramer assembly (44), in addition to the aЈ and bЈ domains, which have been found to fulfill the minimum requirement for function of PDI as a subunit of prolyl 4-hydroxylase (43). (Fig.…”

“…Many experimental data have shown that all four domains cooperate with each other to participate in binding of the protein target (22,43,44), although the bЈ domain contributes to the principal peptide-binding site (45). All four domains have been found to be required for PDI to perform efficiently as a chaperone (22,43,44) and to exhibit maximum catalytic activity (21). The isolated a or aЈ domain can catalyze simple reduction or oxidation reactions of disulfide bonds, but it is not able to catalyze complex isomerizations of proteins containing several disulfide bonds, as native PDI is able to do (46).…”

Functional in Vitro Analysis of the ERO1 Protein and Protein-disulfide Isomerase Pathway

“…Because both the F258W and I272A mutations in the bЈ domain reduce the binding affinity for peptide substrates, we mutated these residues to obtain PDI(sub) (Fig. 4A) (23,24). This mutant showed a marked decrease in the binding affinity for ERO1␣ that was three orders of magnitude weaker than that of wild-type PDI (Fig.…”