Mutations that destabilize the a ' domain of human protein-disulfide isomerase indirectly affect peptide binding

Protein-disulfide isomerase (PDI) is a catalyst of folding of disulfide-bon ded proteins and also a multifunctional polypeptide that acts as the beta-s ubunit in the prolyl 4-hydroxylase alpha(2)beta(2)-tetramer (P4H) and the m icrosomal triglyceride transfer protein alpha beta-dimer. The principal pep tide-binding site of PDI is located in the b' domain, but all domains contr ibute to the binding of misfolded proteins, Mutations in the C-terminal par t of the a' domain have significant effects on the assembly of the P4H tetr amer and other functions of PDI, In this study we have addressed the questi on of whether these mutations in the C-terminal part of the a' domain, whic h affect P4H assembly, also affect peptide binding to PDI. We observed a st rong correlation between P4H assembly competence and peptide binding; mutan ts of PDI that failed to form a functional P4H tetramer were also inactive in peptide binding. However, there was also a correlation between inactivit y in these assays and indicators of conformational disruption, such as prot ease sensitivity. Peptide binding activity could be restored in inactive, p rotease-sensitive mutants by selective proteolytic removal of the mutated a ' domain. Hence we propose that structural changes in the a' domain indirec tly affect peptide binding to the b' domain.