title: Importance of the adaptor (membrane fusion) protein hairpin domain for the functionality of multidrug efflux pumps.
abstract: Drug efflux pumps of Gram-negative bacteria are tripartite export machineries located in the bacterial envelopes contributing to multidrug resistance. Protein structures of all three components have been determined, but the exact interaction sites are still unknown. We could confirm that the hybrid system composed of Pseudomonas aeruginosa channel tunnel OprM and the Escherichia coli inner membrane complex, formed by adaptor protein (membrane fusion protein) AcrA and transporter AcrB of the resistance nodulation cell division (RND) family, is not functional. However, cross-linking experiments show that the hybrid exporter assembles. Exchange of the hairpin domain of AcrA with the corresponding hairpin from adaptor protein MexA of P. aeruginosa restored the functionality. This shows the importance of the MexA hairpin domain for the functional interaction with the OprM channel tunnel. On the basis of these results, we have modeled the interaction of the hairpin domain and the channel tunnel on a molecular level for AcrA and TolC as well as MexA and OprM, respectively. The model of two hairpin docking sites per TolC protomer corresponding with hexameric adaptor proteins was confirmed by disulfide cross-linking experiments. The role of this interaction for functional efflux pumps is discussed.
authors.affiliation: Universität Würzburg, Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany.
authors.forename: Johannes F
authors.initials: JF
authors.lastname: Stegmeier
authors.forename: Georg
authors.initials: G
authors.lastname: Polleichtner
authors.forename: Nicolas
authors.initials: N
authors.lastname: Brandes
authors.forename: Christian
authors.initials: C
authors.lastname: Hotz
authors.forename: Christian
authors.initials: C
authors.lastname: Andersen
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins
chemical_list: D001425:Bacterial Outer Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins