Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD

• DOI: 10.1038/sj.emboj.7600178
• Publication Date: 2004-04-01T11:47:28Z
• Authors: Anna Rozhkova; Christian U Stirn...; Patrick Frei; Ulla Grauschopf; René Brunisholz; Markus G Grütter; Guido Capitani; Rudi Glockshuber
• Keywords: Models, Molecular; 0301 basic medicine; 610 Medicine & health; 10071 Functional Genomics Center Zurich; Genetics and Molecular Biology; Crystallography, X-Ray; Catalysis; Electron Transport; 03 medical and health sciences; 1300 General Biochemistry, Genetics and Molecular Biology; 2400 General Immunology and Microbiology; 1312 Molecular Biology; Escherichia coli; Cysteine; Disulfides; Protein Structure, Quaternary; Molecular Biology; 0303 health sciences; General Immunology and Microbiology; General Neuroscience; Escherichia coli Proteins; 2800 General Neuroscience; Membrane Proteins; 3. Good health; Molecular Weight; Kinetics; General Biochemistry; Periplasm; 570 Life sciences; biology; U7 Systems Biology / Functional Genomics; Oxidoreductases; Oxidation-Reduction; Protein Binding

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.