title: 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 beta-subunit in vertebrate collagen prolyl 4-hydroxylase (C-P4H) alpha(2)beta(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 alphabeta dimer and human alpha(2)beta(2) tetramer formation.
authors.affiliation: Collagen Research Unit, Biocenter Oulu, and Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu FIN-90014, Finland.
authors.forename: Peppi
authors.initials: P
authors.lastname: Koivunen
authors.forename: Kirsi E H
authors.initials: KE
authors.lastname: Salo
authors.forename: Johanna
authors.initials: J
authors.lastname: Myllyharju
authors.forename: Lloyd W
authors.initials: LW
authors.lastname: Ruddock
mesh_terms: D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000201:enzymology*; D002460:Cell Line; D002942:Circular Dichroism; D003094:Collagen / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D017830:Octoxynol / Q000494:pharmacology; D017354:Point Mutation / Q000235:genetics; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012995:Solubility / Q000187:drug effects; D018411:Spodoptera
chemical_list: D021122:Protein Subunits; D017830:Octoxynol; D003094:Collagen; D011393:Procollagen-Proline Dioxygenase; D019286:Endopeptidase K; D019704:Protein Disulfide-Isomerases