title: Peptide binding to protein disulfide isomerase occurs at a site distinct from the active sites.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of South Dakota School of Medicine, Vermillion, 57069.
authors.forename: R
authors.initials: R
authors.lastname: Noiva
authors.forename: R B
authors.initials: RB
authors.lastname: Freedman
authors.forename: W J
authors.initials: WJ
authors.lastname: Lennarz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D003545:Cysteine; D005033:Ethylmaleimide / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007535:Isomerases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases; D051381:Rats; D017207:Rats, Sprague-Dawley; D013879:Thioredoxins / Q000378:metabolism; D014357:Trypsin
chemical_list: D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D010455:Peptides; D002104:Cadmium; D013879:Thioredoxins; D014357:Trypsin; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D003545:Cysteine; D005033:Ethylmaleimide; D003488:Cyanogen Bromide