Peptide binding to protein disulfide isomerase occurs at a site distinct from the active sites.

Protein disulfide isomerase (PDI) is a multifunctional protein resident in the lumen of the rough endoplasmic reticulum that facilitates protein folding via disulfide bond isomerization. Previously we determined that PDI binds a variety of peptides that can be covalently attached to this protein via a photoreactive cross-linker. We have now investigated the relationship between the peptide binding site and the ability of PDI to catalyze disulfide bond isomerization. PDI has two identical sequences, -WCGHCK-, that have been demonstrated to be important in PDI-catalyzed disulfide isomerization. We have found that other proteins containing these thioredoxin-like active site sequences do not bind the photoreactive peptide probes. Moreover, although chemical modification of the 2 cysteines within the thioredoxin-like active site regions completely inhibits PDI-catalyzed disulfide isomerization, these modifications do not affect peptide binding by PDI. Both of these observations suggest that peptide binding occurs at a site other than the putative PDI active sites. To localize the site in PDI at which binding occurs, we used a radiolabeled peptide photoaffinity probe. Peptide fragments generated by cleavage of 125I-peptide-labeled PDI with cyanogen bromide yielded a single 8-kDa polypeptide fragment containing the 125I-labeled peptide site, but neither of the putative catalytic sites of PDI. An 125I-labeled tryptic peptide was generated from this cyanogen bromide fragment and determined by microsequencing to contain residues 451-476 of PDI; this 26-residue peptide is noteworthy because of its extremely high content of acidic amino acids. Based on these findings we conclude that the peptide binding site is located in the COOH-terminal domain of the protein, and it is distinct from the two active sites for PDI-catalyzed disulfide isomerization and from the region of PDI that has estrogen receptor sequence similarity.