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 β-subunit in vertebrate collagen prolyl 4-hydroxylase (C-P4H) α2β2 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 αβ dimer and human α2β2 tetramer formation.