Active-site properties of the oxidized and reduced C-terminal domain of DsbD obtained by NMR spectroscopy.
The periplasmic C-terminal domain of the Escherichia coli DsbD protein (cDsbD) has a thioredoxin fold. The two cysteine residues in the CXXC motif serve as the reductant for the disulfide bond of the N-terminal domain which can in turn act as a reductant for various periplasmic partners. The resulting disulfide bond in cDsbD is reduced via an unknown mechanism by the transmembrane helical domain of the protein. We show by NMR analysis of (13)C, (15)N-labelled cDsbD that the protein is rigid, is stable to extremes of pH and undergoes only localized conformational changes in the vicinity of the CXXC motif, and in adjacent regions of secondary structure, upon undergoing the reduced/oxidized transition. pK(a) values have been determined, using 2D NMR, for the N-terminal cysteine of the CXXC motif, Cys461, as well as for other active-site residues. It is demonstrated using site-directed mutagenesis that the negative charges of the side-chains of Asp455 and Glu468 in the active site contribute to the unusually high pK(a) value, 10.5, of Cys461. This value is higher than expected from knowledge of the reduction potential of cDsbD. In a double mutant of cDsbD, D455N/E468Q, the pK(a) value of Cys461 is lowered to 8.6, a value close to that expected for an unperturbed cysteine residue. The pK(a) value of the second cysteine in wild-type cDsbD, Cys464, is significantly higher than the maximum pH value that was studied (pH 12.2).
Mavridou
Despoina A I
DA
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
Stevens
Julie M
JM
Ferguson
Stuart J
SJ
Redfield
Christina
C
D020816:Amino Acid Motifs; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary
D029968:Escherichia coli Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D010088:Oxidoreductases; C094848:DsbD electron transport protein, E coli; D003545:Cysteine