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Elucidation of the structure and mechanism of the MtrCDE multi drug transporter in Neisseria gonorrhoeae

Zhang, Li (2008) Elucidation of the structure and mechanism of the MtrCDE multi drug transporter in Neisseria gonorrhoeae. Doctoral thesis, Durham University.



The Mtr (Multiple transferable resistance) transport system in Neisseria gonorrhoeae was found to confer the resistance of gonococci to penicillin and structural diverse hydrophobic agents (HAs), such as drugs, dyes, detergents and host-derived compounds (fatty acids and bile salts), as well as some cationic antimicrobial peptides. The mtr operon encodes an energy-dependant efflux pump system and this MtrC-MtrD-MtrE system is negatively regulated by the transcriptional repressor MtrR. The first part of this thesis presents insights into a structural study of the two membrane components, the inner membrane protein MtrD and the outer membrane protein MtrE. MtrD and MtrE have been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method. Unfortunately, due to the difficulty of membrane protein crystallography, the best resolution of the MtrE crystals was only 8A and of the MtrD crystals was 20Å, so the structures of both MtrE and MtrD haven't been solved yet. However, this preliminary X-ray analysis leads to the possibility of solving these structures in the near future. In the mean time, using pull-down assay, growth curve analyses and ITC( Isothermal Titration Calorimetry), we established that the antiporter MtrD, the adaptor MtrC and the outer membrane protein MtrE form a contiguous complex. Assembly of the pump is constitutive, even in absence of substrate. Without the assistance of MtrC, MtrD was unable to export its substrate, Nafcillin, and the efficiency of this assembly could be enhanced by the presence of the outer membrane protein MtrE. MtrD could interact with MtrE independently in pull-down assays, showing they can form a complex even in the absence of MtrC. This behaviour is consistent with the increased hypersensitivity to Vancomycin of the recombinant strain expressing MtrE-MtrD, which indicated that MtrE could be opened by MtrD in the presence of the substrate. However, the interaction between them was not detected by ITC, suggesting this interaction is weak or energetically unfavourable. The a-helical hairpin domain of MtrC was also over expressed and purified to test for its interaction with MtrE or MlrD. Fascinatingly, the cross-linked hairpin formed a hexamer and AFM (Atomic Force Microscopy) studies revealed it arranged into a cylindrical structure. In addition, ITC studies revealed that the hairpin domain could bind to both MtrE and MtrD, suggesting that MtrC might form a channel, one end of which interacts with MtrD and the other with MtrE. Growth curves also showed that the periplasmic hairpin domain could enhance the transport activity of MtrCDE, but couldn't activate the transport of MlrD. indicating it probably works by stabilizeing the open form of MtrE. The expression of mtrCDE is believed to be under the control of a TetR-type transcriptional regulator repressor, mtrR. MtrR foims a dimer but with presence of dsDNA, it forms a tetramer. Fragments of the antimicrobial polypeptide LL-37 were synthesized and tested by titrating to MtrR by ITC. The C-tenninal of LL-37(29-37) is the part that binds to MtrR and N-terminal (I-II) is not. Interestingly, interaction of MtrR with Penicillin G demonstrated for first time that MtrR might work as a ß-lactamase; with its enzyme activity reduced after mutanting His l05 to Tyr, which was reported to be found naturally in penicillin sensitive N, gonorrhoeae strains.

Item Type:Thesis (Doctoral)
Award:Doctor of Philosophy
Thesis Date:2008
Copyright:Copyright of this thesis is held by the author
Deposited On:08 Sep 2011 18:23

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