PEPTIDE BINDING TO PROTEIN DISULFIDE-ISOMERASE OCCURS AT A SITE DISTINCT FROM THE ACTIVE-SITES

Protein disulfide isomerase (PDI) is a multifunctional protein residen t in the lumen of the rough endoplasmic reticulum that facilitates pro tein folding via disulfide bond isomerization. Previously we determine d that PDI binds a variety of peptides that can be covalently attached to this protein via a photoreactive cross-linker. We have now investi gated the relationship between the peptide binding site and the abilit y of PDI to catalyze disulfide bond isomerization. PDI has two identic al sequences, -WCGHCK-, that have been demonstrated to be important in PDI-catalyzed disulfide isomerization. We have found that other prote ins containing these thioredoxin-like active site sequences do not bin d the photoreactive peptide probes. Moreover, although chemical modifi cation of the 2 cysteines within the thioredoxin-like active site regi ons completely inhibits PDI-catalyzed disulfide isomerization, these m odifications do not affect peptide binding by PDI. Both of these obser vations suggest that peptide binding occurs at a site other than the p utative PDI active sites. To localize the site in PDI at which binding occurs, we used a radiolabeled peptide photoaffinity probe. Peptide f ragments generated by cleavage of I-125-peptide-labeled PDI with cyano gen bromide yielded a single 8-kDa polypeptide fragment containing the I-125-labeled peptide site, but neither of the putative catalytic sit es of PDI. An I-125-labeled tryptic peptide was generated from this cy anogen bromide fragment and determined by microsequencing to contain r esidues 451-476 of PDI; this 26-residue peptide is noteworthy because of its extremely high content of acidic amino acids. Based on these fi ndings we conclude that the peptide binding site is located in the COO H-terminal domain of the protein, and it is distinct from the two acti ve sites for PDI-catalyzed disulfide isomerization and from the region of PDI that has estrogen receptor sequence similarity.