CRYSTAL-STRUCTURES OF REDUCED AND OXIDIZED DSBA - INVESTIGATION OF DOMAIN MOTION AND THIOLATE STABILIZATION

Background: The redox proteins that incorporate a thioredoxin fold hav e diverse properties and functions. The bacterial protein-folding fact or DsbA is the most oxidizing of the thioredoxin family. DsbA catalyze s disulfide-bond formation during the folding of secreted proteins, Th e extremely oxidizing nature of DsbA has been proposed to result from either domain motion or stabilizing active-site interactions in the re duced form. In the domain motion model, hinge bending between the two domains of DsbA occurs as a result of redox-related conformational cha nges. Results: We have determined the crystal structures of reduced an d oxidized DsbA in the same crystal form and at the same pH (5.6). The crystal structure of a lower pH form of oxidized DsbA has also been d etermined (pH 5.0). These new crystal structures of DsbA, and the prev iously determined structure of oxidized DsbA at pH 6.5, provide the fo undation for analysis of structural changes that occur upon reduction of the active-site disulfide bond. Conclusions: The structures of redu ced and oxidized DsbA reveal that hinge bending motions do occur betwe en the two domains. These motions are independent of redox state, howe ver, and therefore do not contribute to the energetic differences betw een the two redox states, instead, the observed domain motion is propo sed to be a consequence of substrate binding. Furthermore, DsbA's high ly oxidizing nature is a result of hydrogen bond, electrostatic and he lix-dipole interactions that favour the thiolate over the disulfide at the active site.