Three binding sites in protein-disulfide isomerase cooperate in collagen prolyl 4-hydroxylase tetramer assembly.

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.
Mesh Terms:
Animals, Binding Sites, Caenorhabditis elegans, Cell Line, Circular Dichroism, Collagen, Endopeptidase K, Humans, Octoxynol, Point Mutation, Procollagen-Proline Dioxygenase, Protein Disulfide-Isomerases, Protein Structure, Quaternary, Protein Structure, Tertiary, Protein Subunits, Solubility, Spodoptera
J. Biol. Chem.
Date: Feb. 18, 2005
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