STRUCTURAL-ANALYSIS OF 3 HIS32 MUTANTS OF DSBA - SUPPORT FOR AN ELECTROSTATIC ROLE OF HIS32 IN DSBA STABILITY

DsbA, a 21-kDa protein from Escherichia coli, is a potent oxidizing di sulfide catalyst required for disulfide bond formation in secreted pro teins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and includes a reversible disulfide bond formed from cysteines separated by two residues (Cys3O-Pro31-His32-Cys33). U nlike most protein disulfides, the active-site disulfide of DsbA is hi ghly reactive and the oxidized form of DsbA is much less stable than t he reduced form at physiological pH. His32, one of the two residues be tween the active-site cysteines, is critical to the oxidizing power of DsbA and to the relative instability of the protein in the oxidized f orm. Mutation of this single residue to tyrosine, serine, or leucine r esults in a significant increase in stability (of similar to 5-7 kcal/ mol) of the oxidized His32 variants relative to the oxidized wild-type protein. Despite the dramatic changes in stability, the structures of all three oxidized DsbA His32 Variants are very similar to the wild-t ype oxidized structure, including conservation of solvent atoms near t he active-site residue, Cys3O. These results show that the His32 resid ue does not exert a conformational effect on the structure of DsbA. Th e destabilizing effect of His32 on oxidized DsbA is therefore most lik ely electrostatic in nature.