title: Probing the structure of human protein disulfide isomerase by chemical cross-linking combined with mass spectrometry.
abstract: Protein disulfide-isomerase (PDI) is a four-domain flexible protein that catalyzes the formation of disulfide bonds in the endoplasmic reticulum. Here we have analyzed native PDI purified from human placenta by chemical cross-linking followed by mass spectrometry (CXMS). In addition to PDI the sample contained soluble calnexin and ERp72. Extensive cross-linking was observed within the PDI molecule, both intra- and inter-domain, as well as between the different components in the mixture. The high sensitivity of the analysis in the current experiments, combined with a likely promiscuous interaction pattern of the involved proteins, revealed relatively densely populated cross-link heat maps. The established X-ray structure of the monomeric PDI could be confirmed; however, the dimer as presented in the existing models does not seem to be prevalent in solution as modeling on the observed cross-links revealed new models of dimeric PDI. The observed inter-protein cross-links confirmed the existence of a peptide binding area on calnexin that binds strongly both PDI and ERp72. On the other hand, interaction sites on PDI and ERp72 could not be uniquely identified, indicating a more non-specific interaction pattern.
  
  
  CONCLUSIONS
  The present work demonstrates the use of chemical cross-linking and mass spectrometry (CXMS) for the determination of a solution structure of natural human PDI and its interaction with the chaperones ERp72 and calnexin. The data shows that the dimeric structure of PDI may be more diverse than indicated by present models. We further observe that the temperature influences the cross-linking pattern of PDI, but this does not influence the overall folding pattern of the molecule.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: Li
authors.initials: L
authors.lastname: Peng
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: Morten Ib
authors.initials: MI
authors.lastname: Rasmussen
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: Anna
authors.initials: A
authors.lastname: Chailyan
authors.affiliation: Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Houen
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark. Electronic address: php@bmb.sdu.dk.
authors.forename: Peter
authors.initials: P
authors.lastname: Højrup
mesh_terms: D018360:Crystallography, X-Ray; D005260:Female; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D011257:Pregnancy Proteins / Q000737:chemistry* / Q000302:isolation & purification; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000302:isolation & purification; D017434:Protein Structure, Tertiary
chemical_list: D011257:Pregnancy Proteins; D019704:Protein Disulfide-Isomerases
keywords: BS3; Calnexin; Cross-linking; ERp72; Placenta; Protein structure