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Evidence for the assembly of a bacterial tripartite multidrug pump with a stoichiometry of 3:6:3.

Janganan, T.K. and Bavro, V.N. and Zhang, L. and Matak-Vinkovic, D. and Barrera, N.P. and Venien-Bryan, C. and Robinson, C.V. and Borges-Walmsley, M.I. and Walmsley, A.R. (2011) 'Evidence for the assembly of a bacterial tripartite multidrug pump with a stoichiometry of 3:6:3.', Journal of biological chemistry., 286 (30). pp. 26900-26912.

Abstract

The multiple transferable resistance (mTR) pump from Neisseria gonorrhoeae MtrCDE multidrug pump is assembled from the inner and outer membrane proteins MtrD and MtrE and the periplasmic membrane fusion protein MtrC. Previously we established that while there is a weak interaction of MtrD and MtrE, MtrC binds with relatively high affinity to both MtrD and MtrE. MtrD conferred antibiotic resistance only when it was expressed with MtrE and MtrC, suggesting that these proteins form a functional tripartite complex in which MtrC bridges MtrD and MtrE. Furthermore, we demonstrated that MtrC interacts with an intraprotomer groove on the surface of MtrE, inducing channel opening. However, a second groove is apparent at the interface of the MtrE subunits, which might also be capable of engaging MtrC. We have now established that MtrC can be cross-linked to cysteines placed in this interprotomer groove and that mutation of residues in the groove impair the ability of the pump to confer antibiotic resistance by locking MtrE in the closed channel conformation. Moreover, MtrE K390C forms an intermolecular disulfide bond with MtrC E149C locking MtrE in the open channel conformation, suggesting that a functional salt bridge forms between these residues during the transition from closed to open channel conformations. MtrC forms dimers that assemble into hexamers, and electron microscopy studies of single particles revealed that these hexamers are arranged into ring-like structures with an internal aperture sufficiently large to accommodate the MtrE trimer. Cross-linking of single cysteine mutants of MtrC to stabilize the dimer interface in the presence of MtrE, trapped an MtrC-MtrE complex with a molecular mass consistent with a stoichiometry of 3:6 (MtrE3MtrC6), suggesting that dimers of MtrC interact with MtrE, presumably by binding to the two grooves. As both MtrE and MtrD are trimeric, our studies suggest that the functional pump is assembled with a stoichiometry of 3:6:3.

Item Type:Article
Keywords:Antibiotics, Bacteria, Membrane Proteins, Multidrug Transporters, Protein Assembly, Neisseria gonorrhoeae.
Full text:PDF - Published Version (1827Kb)
Status:Peer-reviewed
Publisher Web site:http://dx.doi.org/10.1074/jbc.M111.246595
Publisher statement:Full-text available under a Creative Commons Attribution Non-Commercial License.
Record Created:01 Jun 2012 17:35
Last Modified:06 Jun 2012 10:09

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