title: Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase.
abstract: Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b' domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b' domain and show the existence of an intradomain linker between the b' and a' domains. We have expressed the recombinant b' domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b' domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect.
authors.affiliation: Biocenter Oulu and Department of Biochemistry, University of Oulu, Finland.
authors.forename: Annamari
authors.initials: A
authors.lastname: Pirneskoski
authors.forename: Peter
authors.initials: P
authors.lastname: Klappa
authors.forename: Mario
authors.initials: M
authors.lastname: Lobell
authors.forename: Richard A
authors.initials: RA
authors.lastname: Williamson
authors.forename: Lee
authors.initials: L
authors.lastname: Byrne
authors.forename: Heli I
authors.initials: HI
authors.lastname: Alanen
authors.forename: Kirsi E H
authors.initials: KE
authors.lastname: Salo
authors.forename: Kari I
authors.initials: KI
authors.lastname: Kivirikko
authors.forename: Robert B
authors.initials: RB
authors.lastname: Freedman
authors.forename: Lloyd W
authors.initials: LW
authors.lastname: Ruddock
mesh_terms: D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D015153:Blotting, Western; D002384:Catalysis; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013879:Thioredoxins / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D010455:Peptides; D011994:Recombinant Proteins; D013879:Thioredoxins; D019704:Protein Disulfide-Isomerases; D010100:Oxygen