title: Sequential mechanism of assembly of multidrug efflux pump AcrAB-TolC.
abstract: Multidrug efflux pumps adversely affect both the clinical effectiveness of existing antibiotics and the discovery process to find new ones. In this study, we reconstituted and characterized by surface plasmon resonance the assembly of AcrAB-TolC, the archetypal multidrug efflux pump from Escherichia coli. We report that the periplasmic AcrA and the outer membrane channel TolC assemble high-affinity complexes with AcrB transporter independently from each other. Antibiotic novobiocin and MC-207,110 inhibitor bind to the immobilized AcrB but do not affect interactions between components of the complex. In contrast, DARPin inhibits interactions between AcrA and AcrB. Mutational opening of TolC channel decreases stability of interactions and promotes disassembly of the complex. The conformation of the membrane proximal domain of AcrA is critical for the formation of AcrAB-TolC and could be targeted for the development of new inhibitors.
authors.affiliation: Department of Chemistry and Biochemistry, Stephenson Life Science Research Center, University of Oklahoma, Norman, OK 73019, USA.
authors.forename: Elena B
authors.initials: EB
authors.lastname: Tikhonova
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Yamada
authors.forename: Helen I
authors.initials: HI
authors.lastname: Zgurskaya
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055767:Immobilized Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D050356:Lipid Metabolism; D008074:Lipoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D020349:Surface Plasmon Resonance
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D055767:Immobilized Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083497:AcrA protein, E coli; C083496:AcrB protein, E coli; C073126:tolC protein, E coli