Evidence for the assembly of a bacterial tripartite multidrug pump with a stoichiometry of 3:6:3.
Authors
Janganan, Thamarai K.Bavro, Vassiliy N
Zhang, Li
Matak-Vinkovic, Dijana
Barrera, Nelson P.
Venien-Bryan, Catherine
Robinson, Carol V.
Borges-Walmsley, Maria Ines
Walmsley, Adrian R.
Affiliation
University of DurhamUniversity of Oxford
University of Cambridge
Pontificia Universidad Catolica de Chile
Issue Date
2011-07-29
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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 (MtrE(3)MtrC(6)), 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.Citation
Janganan TK, Bavro VN, Zhang L, Matak-Vinkovic D, Barrera NP, Venien-Bryan C, Robinson CV, Borges-Walmsley MI, Walmsley AR (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.26900-12.Journal
Journal of Biological ChemistryPubMed ID
21610073Additional Links
http://www.jbc.org/content/286/30/26900.shortType
ArticleLanguage
enISSN
0021-9258ae974a485f413a2113503eed53cd6c53
10.1074/jbc.M111.246595
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