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

  • Thamarai Janganan
    ,
  • Vassiliy N. Bavro
    ,
  • Li Zhang
    ,
  • Dijana Matak-Vinkovic
    ,
  • Nelson P. Barrera
    ,
  • Catherine Venien-Bryan
  • University of Oxford
    ,
  • Durham University
    ,
  • University of Cambridge
    ,
  • Pontificia Universidad Católica de Chile
Research Output: Contribution to journal Article Peer-review

Open access

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 (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.

Publication Information

Output type

Research Output: Contribution to journal Article Peer-review

Original language

English

Pages from-to (Number of pages)

Pages 26900-12

Journal (Volume, Issue Number)

Journal of Biological Chemistry (Volume 286, Issue 30)

Publication milestones

  • Published - 29/07/2011

Publication status

Published - 29/07/2011

ISSN

0021-9258

External Publication IDs

  • handle.net: 10547/622748
  • Scopus: 79960663093

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