On the role of the cis-proline residue in the active site of DsbA

In addition to the Cys-Xaa-Xaa-Cys motif at position 30-33, DsbA, the essen tial catalyst for disulfide bond formation in the bacterial periplasm share s with other oxidoreductases of the thioredoxin family a cis-proline in pro ximity of the active site residues. In the variant DsbA(P151A), this residu e has been changed to an alanine, an almost isosteric residue which is not disposed to adopt the cis conformation. The substitution strongly destabili zed the structure of DsbA, as determined by the decrease in the free energy of folding. The pK(a) of the thiol of Cys30 was only marginally decreased. Although in vivo the variant appeared to be correctly oxidized, it exhibit ed an activity less than half that of the wild-type enzyme with respect to the folding of alkaline phosphatase, used as a reporter of the disulfide bo nd formation in the periplasm. DsbA(P151A) crystallized in a different crys tal form from the wild-type protein, in space group P2(1) with six molecule s in the asymmetric unit. Its X-ray structure was determined to 2.8 Angstro m resolution. The most significant conformational changes occurred at the a ctive site. The loop 149-152 adopted a new backbone conformation with Ala15 1 in a trans conformation. This rearrangement resulted in the loss of van d er Waals interactions between this loop and the disulfide bond. His32 from the Cys-Xaa-Xaa-Cys sequence presented in four out of six molecules in the asymmetric unit a gauche-conformation not observed in the wild-type protein . The X-ray structure and folding studies on DsbA(P151A) were consistent wi th the cis-proline playing a major role in the stabilization of the protein . A role for the positioning of the substrate is discussed. These important properties for the enzyme function might explain the conservation of this residue in DsbA and related proteins possessing the thioredoxin fold.