Du, Dijun (2006) Elucidation of the structure and molecular mechanism of the tripartite multidrug efflux pumps in the Gram-negative pathogens: Vibrio cholerae and Neisseria gonorrhoeae. Doctoral thesis, Durham University.
In bacteria, multidrug efflux systems have been identified as significant determinants of resistance recently. These resistance pumps are widely distributed in bacterial species and many pathogenic bacteria posses them, which play an important role in their intrinsic and acquired multidrug resistances. The RND and MATE family transporters have also been shown to be involved in the pathogenicity of bacteria. Knowledge of the structure and mechanism of these transporter proteins would be exceedingly useful in the design of inhibitors. In Gram-negative bacteria, multidrug resistance is conferred in part by the tripartite multidrug efflux pumps that are composed of an inner membrane transport protein, a membrane fusion protein and an outer membrane protein. One such tripartite pump, VceCAB of Vibrio cholerae, is composed of an inner membrane H(^+)-antiporter VceB, a membrane fusion protein VceA and an outer membrane channel VceC. To investigate the role of this pump in the multidrug resistance of Vibrio cholerae, we have characterized functionally and structurally the three components of the VceCAB pump and the regulator VceR. The crystal structure of VceC was determined at 1.8 Â resolutions. Despite the very low degree of sequence identity between them, VceC shares the same overall architecture as TolC, consisting of three domains: the ß-domain, the α-domain and the equatorial domain. The trimeric VceC packs in laminar sheets in the crystal that resemble membranes. Like TolC, the α-barrel of the VceC channel at the periplasmic end is closed through the packing interactions of coiled-coil helices, but the residues that maintain the closed state of the channels of VceC and TolC are different. The ß-barrel region of VceC is also closed, whereas the ß-barrel region of TolC is open to the extracellular medium. The channel interior of VceC is generally electronegative and contains two rings of clusters negative charge. The ring made by residues Glu(^397) and Glu(^303) is conserved in OprM, but is not in TolC. Mutagenesis assay of this negative charged ring indicated its functional role during transport. The optimal desolvation area (ODA) on the surface of VceC is different from that of TolC, suggesting distinct architectures of VceC-based and TolC-based tripartite pumps. Sub-cellular fractionation of cells expressing full length and truncated VceA suggested that VceA is anchored to the IM via a transmembrane helix. Analytical gel filtration chromatography experiments revealed that the periplasmic domain of VceA that was expressed in the periplasm of E.coil forais a trimer, which could represent its oligomeric state in the VceCAB pump. The three components of tripartite pumps are easy to dissociate in vitro, making it difficult to co-crystallize them. We overproduced the protein complex in which VceA (12-406) is in complex with the VceB-VceA fusion protein. This complex was stable during purification, which could provide an invaluable way for co-crystallization of these two components of the VceCAB pump. An analytical gel filtration and DLS experiments indicated that the basic functional unit of VceR is a dimer; the binding of substrate СССР to VceR has been determined to occur with a Hill coefficient of about four, and thus each VceR dimer binds four СССР molecules. This stoichiometry of drug/VceR-subunit is different from that of other transcriptional regulators in the TetR/CamR family. There are differences between MtrD and other RND family multidrug efflux pumps. The knowledge of difference will be important for understanding the mechanism of these family transporters. In this study, we successfully overexpressed, purified and crystallized MtrD. The resolution of MtrD crystals was optimised to 7-10 Å at present.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||09 Sep 2011 09:51|