Structural studies of the electron transport proteins DsbD and Rv2874

Abstract

The research in this thesis focuses on DsbD, the Escherichia coli integral membrane electron transport protein and a related protein, Rv2874, from Mycobacterium tuberculosis. During protein disulfide bond formation it is possible for disulfide bonds to be formed between cysteine residues which are distant in the final three-dimensional structure of a protein. These disulfide bonds may trap a protein in a non-native state rendering the protein inactive. Within the periplasm of E. coli the disulfide bond rearrangement pathway uses electrons generated in the cytoplasm of the cell to break these disulfide bonds allowing the protein to refold. The integral membrane electron transporter DsbD, a member of the CcdA family of electron transport proteins, shuttles electrons across the inner membrane of the cell, maintaining the disulfide bond isomerases, DsbC and DsbG, in the active reduced form. To investigate the mechanism of electron transport by DsbD and the CcdA-family of electron transporters, studies have been undertaken to determine the structure of DsbD and the C-terminal domain of the related protein Rv2874 from the Gram-positive bacteria M. tuberculosis, the causative agent for tuberculosis. The structure of the N-terminal domain of DsbD (DsbDa) has been determined at a resolution of 1.9 A and reveals a new type of thiol-oxidoreductase with an Ig-fold. The active site of DsbDa is sequestered from the surrounding environment by the presence of a cap structure which regulates the accessibility to the active site cysteine residues. Substantial progress has also been made towards the determination of the structure of full-length DsbD. A robust purification protocol has been developed and the stability of DsbD in a range of detergents has been examined. Crystallisation trials of DsbD in various detergents have resulted in three conditions which produced crystals. The structure of the C-terminal domain of the related CcdA-like protein Rv2874 from Mycobacterium tuberculosis has been determined at a resolution of 1.9 A. Along with the expected thioredoxin fold, the structure reveals an unexpected second domain with similarity to carbohydrate binding modules suggesting a broadening in the functional repertoire of CcdA-like proteins and a possible role for Rv2874 in carbohydrate processing.