title: Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD.
abstract: DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic disulfide isomerase DsbC. DsbD contains two periplasmically oriented domains at the N- and C-terminus (nDsbD and cDsbD) that are connected by a central transmembrane (TM) domain. Each domain contains a pair of cysteines that are essential for catalysis. Here, we show that Cys109 and Cys461 form a transient interdomain disulfide bond between nDsbD and cDsbD in the reaction cycle of DsbD. We solved the crystal structure of this catalytic intermediate at 2.85 A resolution, which revealed large relative domain movements in DsbD as a consequence of a strong overlap between the surface areas of nDsbD that interact with DsbC and cDsbD. In addition, we have measured the kinetics of all functional and nonfunctional disulfide exchange reactions between redox-active, periplasmic proteins and protein domains from the oxidative DsbA/B and the reductive DsbC/D pathway. We show that both pathways are separated by large kinetic barriers for nonfunctional disulfide exchange between components from different pathways.
authors.affiliation: Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland.
authors.forename: Anna
authors.initials: A
authors.lastname: Rozhkova
authors.forename: Christian U
authors.initials: CU
authors.lastname: Stirnimann
authors.forename: Patrick
authors.initials: P
authors.lastname: Frei
authors.forename: Ulla
authors.initials: U
authors.lastname: Grauschopf
authors.forename: René
authors.initials: R
authors.lastname: Brunisholz
authors.forename: Markus G
authors.initials: MG
authors.lastname: Grütter
authors.forename: Guido
authors.initials: G
authors.lastname: Capitani
authors.forename: Rudi
authors.initials: R
authors.lastname: Glockshuber
mesh_terms: D002384:Catalysis; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D004579:Electron Transport; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary
chemical_list: D004220:Disulfides; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010088:Oxidoreductases; C094848:DsbD electron transport protein, E coli; D003545:Cysteine