title: Kinetic control of TolC recruitment by multidrug efflux complexes.
abstract: In Gram-negative pathogens, multidrug efflux pumps that provide clinically significant levels of antibiotic resistance function as three-component complexes. They are composed of the inner membrane transporters belonging to one of three superfamilies of proteins, RND, ABC, or MF; periplasmic proteins belonging to the membrane fusion protein (MFP) family; and outer membrane channels exemplified by the Escherichia coli TolC. The three-component complexes span the entire two-membrane envelope of Gram-negative bacteria and expel toxic molecules from the cytoplasmic membrane to the medium. The architecture of these complexes is expected to vary significantly because of the structural diversity of the inner membrane transporters. How the three-component pumps are assembled, their architecture, and their dynamics remain unclear. In this study, we reconstituted interactions and compared binding kinetics of the E. coli TolC with AcrA, MacA, and EmrA, the periplasmic MFPs that function in multidrug efflux with transporters from the RND, ABC, and MF superfamilies, respectively. By using surface plasmon resonance, we demonstrate that TolC interactions with MFPs are highly dynamic and sensitive to pH. The affinity of TolC to MFPs decreases in the order MacA > EmrA > AcrA. We further show that MFPs are prone to oligomerization, but differ dramatically from each other in oligomerization kinetics and stability of oligomers. The propensity of MFPs to oligomerize correlates with the stability of MFP-TolC complexes and structural features of inner membrane transporters. We propose that recruitment of TolC by various MFPs is determined not only by kinetics of MFP-TolC interactions but also by oligomerization kinetics of MFPs and pH.
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Room 208, Norman, OK 73019, USA.
authors.forename: Elena B
authors.initials: EB
authors.lastname: Tikhonova
authors.forename: Vishakha
authors.initials: V
authors.lastname: Dastidar
authors.forename: Valentin V
authors.initials: VV
authors.lastname: Rybenkov
authors.forename: Helen I
authors.initials: HI
authors.lastname: Zgurskaya
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D020349:Surface Plasmon Resonance
chemical_list: D018528:ATP-Binding Cassette Transporters; C083497:AcrA protein, E coli; D001425:Bacterial Outer Membrane Proteins; C489681:EmrA protein, E coli; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C436312:MacA protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli