title: Stoichiometry of the MexA-OprM binding, as investigated by blue native gel electrophoresis.
abstract: Multidrug resistance has become a serious concern in the treatment of bacterial infections. A prominent role is ascribed to the active efflux of xenobiotics out of the bacteria by a tripartite protein machinery. The mechanism of drug extrusion is rather well understood, thanks to the X-ray structures obtained for the Escherichia coli TolC/AcrA/AcrB model system and the related Pseudomonas aeruginosa OprM/MexA/MexB. However, many questions remain unresolved, in particular the stoichiometry of the efflux pump assembly. On the basis of blue native polyacrylamide gel electrophoresis (BN-PAGE) (Wittig et al., Nat. Protoc. 2006, 1, 418-428), we analyzed the binding stoichiometry of both palmitylated and non-palmitylated MexA with the cognate partner OprM trimer at different ratios and detergent conditions. We found that β-octyl glucopyranoside (β-OG) detergent was not suitable for this technique. Then we proved that MexA has to be palmitylated in order to stabilized the complex formation with OprM. Finally, we provided evidence for a two by two (2, 4, 6, or upper) binding of palmitylated MexA per trimer of OprM.
authors.affiliation: Laboratoire de Cristallographie et RMN Biologiques, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Paris, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Ferrandez
authors.forename: Laura
authors.initials: L
authors.lastname: Monlezun
authors.forename: Gilles
authors.initials: G
authors.lastname: Phan
authors.forename: Houssain
authors.initials: H
authors.lastname: Benabdelhak
authors.forename: Philippe
authors.initials: P
authors.lastname: Benas
authors.forename: Nathalie
authors.initials: N
authors.lastname: Ulryck
authors.forename: Pierre
authors.initials: P
authors.lastname: Falson
authors.forename: Arnaud
authors.initials: A
authors.lastname: Ducruix
authors.forename: Martin
authors.initials: M
authors.lastname: Picard
authors.forename: Isabelle
authors.initials: I
authors.lastname: Broutin
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D018432:Drug Resistance, Multiple; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D010169:Palmitic Acids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding
chemical_list: D001425:Bacterial Outer Membrane Proteins; D026901:Membrane Transport Proteins; C118598:MexA protein, Pseudomonas aeruginosa; D046912:Multiprotein Complexes; C402263:OprM protein, Pseudomonas aeruginosa; D010169:Palmitic Acids