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

2.50
Hdl Handle:
http://hdl.handle.net/10547/622748
Title:
Evidence for the assembly of a bacterial tripartite multidrug pump with a stoichiometry of 3:6:3.
Authors:
Janganan, Thamarai K.; Bavro, Vassiliy N ( 0000-0003-2330-8924 ) ; Zhang, Li; Matak-Vinkovic, Dijana; Barrera, Nelson P.; Venien-Bryan, Catherine; Robinson, Carol V.; Borges-Walmsley, Maria Ines; Walmsley, Adrian R. ( 0000-0002-5136-0511 )
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.
Affiliation:
University of Durham; University of Oxford; University of Cambridge; Pontificia Universidad Catolica de Chile
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.
Publisher:
American Society for Biochemistry and Molecular Biology
Journal:
Journal of Biological Chemistry
Issue Date:
29-Jul-2011
URI:
http://hdl.handle.net/10547/622748
DOI:
10.1074/jbc.M111.246595
PubMed ID:
21610073
Additional Links:
http://www.jbc.org/content/286/30/26900.short
Type:
Article
Language:
en
ISSN:
0021-9258
Appears in Collections:
Biomedical and biological science

Full metadata record

DC FieldValue Language
dc.contributor.authorJanganan, Thamarai K.en
dc.contributor.authorBavro, Vassiliy Nen
dc.contributor.authorZhang, Lien
dc.contributor.authorMatak-Vinkovic, Dijanaen
dc.contributor.authorBarrera, Nelson P.en
dc.contributor.authorVenien-Bryan, Catherineen
dc.contributor.authorRobinson, Carol V.en
dc.contributor.authorBorges-Walmsley, Maria Inesen
dc.contributor.authorWalmsley, Adrian R.en
dc.date.accessioned2018-06-20T10:59:58Z-
dc.date.available2018-06-20T10:59:58Z-
dc.date.issued2011-07-29-
dc.identifier.citationJanganan 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.en
dc.identifier.issn0021-9258-
dc.identifier.pmid21610073-
dc.identifier.doi10.1074/jbc.M111.246595-
dc.identifier.urihttp://hdl.handle.net/10547/622748-
dc.description.abstractThe 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.en
dc.language.isoenen
dc.publisherAmerican Society for Biochemistry and Molecular Biologyen
dc.relation.urlhttp://www.jbc.org/content/286/30/26900.shorten
dc.rightsGreen - can archive pre-print and post-print or publisher's version/PDF-
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/*
dc.subjectmultidrug transportersen
dc.subjectNeisseria gonorrhoeaeen
dc.subjectmultidrug pumpen
dc.titleEvidence for the assembly of a bacterial tripartite multidrug pump with a stoichiometry of 3:6:3.en
dc.typeArticleen
dc.contributor.departmentUniversity of Durhamen
dc.contributor.departmentUniversity of Oxforden
dc.contributor.departmentUniversity of Cambridgeen
dc.contributor.departmentPontificia Universidad Catolica de Chileen
dc.identifier.journalJournal of Biological Chemistryen
dc.date.updated2018-06-20T09:02:40Z-
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