scholarly journals Covalently Linked Trimer of the AcrB Multidrug Efflux Pump Provides Support for the Functional Rotating Mechanism

2008 ◽  
Vol 191 (6) ◽  
pp. 1729-1737 ◽  
Author(s):  
Yumiko Takatsuka ◽  
Hiroshi Nikaido
Keyword(s):  
Conformational Changes ◽  
Transmembrane Domain ◽  
Efflux Pump ◽  
Polyacrylamide Gel Electrophoresis ◽  
Residual Activity ◽  
Salt Bridge ◽  
Relay Network ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Intact Cells

ABSTRACT Escherichia coli AcrB is a proton motive force-dependent multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export (functional rotation hypothesis). In this study, we devised a way to test this hypothesis by creating a giant gene in which three acrB sequences were connected together through short linker sequences. The “linked-trimer” AcrB was expressed well in the inner membrane fraction of ΔacrB ΔrecA strains, as a large protein of ∼300 kDa which migrated at the same rate as the wild-type AcrB trimer in native polyacrylamide gel electrophoresis. The strain expressing the linked-trimer AcrB showed resistance to some toxic compounds that was sometimes even higher than that of the cells expressing the monomeric AcrB, indicating that the linked trimer functions well in intact cells. When we inactivated only one of the three protomeric units in the linked trimer, either with mutations in the salt bridge/H-bonding network (proton relay network) in the transmembrane domain or by disulfide cross-linking of the external cleft in the periplasmic domain, the entire trimeric complex was inactivated. However, some residual activity was seen, presumably as a result of random recombination of monomeric fragments (produced by protease cleavage or by transcriptional/translational truncation). These observations provide strong biochemical evidence for the functionally rotating mechanism of AcrB pump action. The linked trimer will be useful for further biochemical studies of mechanisms of transport in the future.

scholarly journals Conformation of the AcrB Multidrug Efflux Pump in Mutants of the Putative Proton Relay Pathway

2006 ◽  
Vol 188 (20) ◽  
pp. 7290-7296 ◽  
Author(s):  
Chih-Chia Su ◽  
Ming Li ◽  
Ruoyu Gu ◽  
Yumiko Takatsuka ◽  
Gerry McDermott ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Transmembrane Domain ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Mutant Proteins ◽  
X Ray ◽  
Proton Relay ◽  
Initial Binding

ABSTRACT We previously reported the X-ray structures of wild-type Escherichia coli AcrB, a proton motive force-dependent multidrug efflux pump, and its N109A mutant. These structures presumably reflect the resting state of AcrB, which can bind drugs. After ligand binding, a proton may bind to an acidic residue(s) in the transmembrane domain, i.e., Asp407 or Asp408, within the putative network of electrostatically interacting residues, which also include Lys940 and Thr978, and this may initiate a series of conformational changes that result in drug expulsion. Herein we report the X-ray structures of four AcrB mutants, the D407A, D408A, K940A, and T978A mutants, in which the structure of this tight electrostatic network is expected to become disrupted. These mutant proteins revealed remarkably similar conformations, which show striking differences from the previously known conformations of the wild-type protein. For example, the loop containing Phe386 and Phe388, which play a major role in the initial binding of substrates in the central cavity, becomes prominently extended into the center of the cavity, such that binding of large substrate molecules may become difficult. We believe that this new conformation may mimic, at least partially, one of the transient conformations of the transporter during the transport cycle.

scholarly journals Threonine-978 in the Transmembrane Segment of the Multidrug Efflux Pump AcrB of Escherichia coli Is Crucial for Drug Transport as a Probable Component of the Proton Relay Network

2006 ◽  
Vol 188 (20) ◽  
pp. 7284-7289 ◽  
Author(s):  
Yumiko Takatsuka ◽  
Hiroshi Nikaido
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Bonding ◽  
Conformational Changes ◽  
Drug Transport ◽  
Efflux Pump ◽  
Relay Network ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Alanine Scanning Mutagenesis ◽  
Proton Relay

ABSTRACT Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals and expels them from cells. It is a proton antiporter belonging to the resistance-nodulation-division (RND) superfamily. Asp407, Asp408, Lys940, and Arg971 in transmembrane (TM) helices of this transporter have been identified as essential amino acid residues that probably function as components of the proton relay system. In this study, we identified a novel residue in TM helix 11, Thr978, as an essential residue by alanine scanning mutagenesis. Its location close to Asp407 suggests that it is also a component of the proton translocation pathway, a prediction confirmed by the similar conformations adopted by T978A, D407A, D408A, and K940A mutant proteins (see the accompanying paper). Sequence alignment of 566 RND transporters showed that this threonine residue is conserved in about 96% of cases. Our results suggest the hypotheses that Thr978 functions through hydrogen bonding with Asp407 and that protonation of the latter alters the salt bridging and hydrogen bonding pattern in the proton relay network, thus initiating a series of conformational changes that ultimately result in drug extrusion.

scholarly journals A Periplasmic Drug-Binding Site of the AcrB Multidrug Efflux Pump: a Crystallographic and Site-Directed Mutagenesis Study

2005 ◽  
Vol 187 (19) ◽  
pp. 6804-6815 ◽  
Author(s):  
Edward W. Yu ◽  
Julio R. Aires ◽  
Gerry McDermott ◽  
Hiroshi Nikaido
Keyword(s):  
Binding Site ◽  
Transmembrane Domain ◽  
Efflux Pump ◽  
Drug Binding ◽  
Site Directed Mutagenesis ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Central Cavity ◽  
Intact Cells ◽  
Drug Binding Site

ABSTRACT The Escherichia coli AcrB multidrug efflux pump is a membrane protein that recognizes many structurally dissimilar toxic compounds. We previously reported the X-ray structures of four AcrB-ligand complexes in which the ligands were bound to the wall of the extremely large central cavity in the transmembrane domain of the pump. Genetic studies, however, suggested that discrimination between the substrates occurs mainly in the periplasmic domain rather than the transmembrane domain of the pump. We here describe the crystal structures of the AcrB mutant in which Asn109 was replaced by Ala, with five structurally diverse ligands, ethidium, rhodamine 6G, ciprofloxacin, nafcillin, and Phe-Arg-β-naphthylamide. The ligands bind not only to the wall of central cavity but also to a new periplasmic site within the deep external depression formed by the C-terminal periplasmic loop. This depression also includes residues identified earlier as being important in the specificity. We show here that conversion into alanine of the Phe664, Phe666, or Glu673 residue in the periplasmic binding site produced significant decreases in the MIC of most agents in the N109A background. Furthermore, decreased MICs were also observed when these residues were mutated in the wild-type AcrB background, although the effects were more modest. The MIC data were also confirmed by assays of ethidium influx rates in intact cells, and our results suggest that the periplasmic binding site plays a role in the physiological process of drug efflux.

scholarly journals Cross-Linked Complex between Oligomeric Periplasmic Lipoprotein AcrA and the Inner-Membrane-Associated Multidrug Efflux Pump AcrB from Escherichia coli

2000 ◽  
Vol 182 (15) ◽  
pp. 4264-4267 ◽  
Author(s):  
Helen I. Zgurskaya ◽  
Hiroshi Nikaido
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Intact Cells ◽  
Inner Membrane ◽  
Oligomeric Form ◽  
Chemical Cross Linking ◽  
Multidrug Efflux System

ABSTRACT In Escherichia coli, the intrinsic levels of resistance to multiple antimicrobial agents are produced through expression of the three-component multidrug efflux system AcrAB-TolC. AcrB is a proton-motive-force-dependent transporter located in the inner membrane, and AcrA and TolC are accessory proteins located in the periplasm and the outer membrane, respectively. In this study, these three proteins were expressed separately, and the interactions between them were analyzed by chemical cross-linking in intact cells. We show that AcrA protein forms oligomers, most probably trimers. In this oligomeric form, AcrA interacts specifically with AcrB transporter independently of substrate and TolC.

scholarly journals Bile Salts Modulate Expression of the CmeABC Multidrug Efflux Pump in Campylobacter jejuni

2005 ◽  
Vol 187 (21) ◽  
pp. 7417-7424 ◽  
Author(s):  
Jun Lin ◽  
Cédric Cagliero ◽  
Baoqing Guo ◽  
Yi-Wen Barton ◽  
Marie-Christine Maurel ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Conformational Changes ◽  
Bile Salts ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Dependent Manner ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Altered Expression ◽  
Bile Resistance

ABSTRACT CmeABC, a multidrug efflux pump, is involved in the resistance of Campylobacter jejuni to a broad spectrum of antimicrobial agents and is essential for Campylobacter colonization in animal intestine by mediating bile resistance. Previously, we have shown that expression of this efflux pump is under the control of a transcriptional repressor named CmeR. Inactivation of CmeR or mutation in the cmeABC promoter (P cmeABC ) region derepresses cmeABC, leading to overexpression of this efflux pump. However, it is unknown if the expression of cmeABC can be conditionally induced by the substrates it extrudes. In this study, we examined the expression of cmeABC in the presence of various antimicrobial compounds. Although the majority of the antimicrobials tested did not affect the expression of cmeABC, bile salts drastically elevated the expression of this efflux operon. The induction was observed with both conjugated and unconjugated bile salts and was in a dose- and time-dependent manner. Experiments using surface plasmon resonance demonstrated that bile salts inhibited the binding of CmeR to P cmeABC , suggesting that bile compounds are inducing ligands of CmeR. The interaction between bile salts and CmeR likely triggers conformational changes in CmeR, resulting in reduced binding affinity of CmeR to P cmeABC . Bile did not affect the transcription of cmeR, indicating that altered expression of cmeR is not a factor in bile-induced overexpression of cmeABC. In addition to the CmeR-dependent induction, some bile salts (e.g., taurocholate) also activated the expression of cmeABC by a CmeR-independent pathway. Consistent with the elevated production of CmeABC, the presence of bile salts in culture media resulted in increased resistance of Campylobacter to multiple antimicrobials. These findings reveal a new mechanism that modulates the expression of cmeABC and further support the notion that bile resistance is a natural function of CmeABC.

The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump

2015 ◽  
Vol 396 (9-10) ◽  
pp. 1073-1082 ◽  
Author(s):  
Dijun Du ◽  
Jarrod Voss ◽  
Zhao Wang ◽  
Wah Chiu ◽  
Ben F. Luisi
Keyword(s):  
Antimicrobial Agents ◽  
Transmembrane Domain ◽  
Efflux Pump ◽  
Cell Envelope ◽  
Toxic Substances ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Transporter Family ◽  
Wide Range ◽  
Rnd Transporter

Abstract Microorganisms encode several classes of transmembrane molecular pumps that can expel a wide range of chemically distinct toxic substances. These machines contribute to the capacity of the organisms to withstand harsh environments, and they help to confer resistance against clinical antimicrobial agents. In Gram-negative bacteria, some of the pumps comprise tripartite assemblies that actively transport drugs and other harmful compounds across the cell envelope. We describe recent structural and functional data that have provided insights into the architecture and transport mechanism of the AcrA-AcrB-TolC pump of Escherichia coli. This multidrug efflux pump is powered by proton electrochemical gradients through the activity of AcrB, a member of the resistance/nodulation/cell division (RND) transporter family. Crystallographic data reveal how the small protein AcrZ binds to AcrB in a concave surface of the transmembrane domain, and we discuss how this interaction may affect the efflux activities of the transporter.

scholarly journals Interactions of a bacterial RND transporter with a transmembrane small protein in a lipid environment

2019 ◽  
Author(s):  
Dijun Du ◽  
Arthur Neuberger ◽  
Mona Wu Orr ◽  
Catherine E. Newman ◽  
Pin-Chia Hsu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Sensitivity ◽  
Efflux Pump ◽  
Binding Pocket ◽  
Drug Binding ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Small Protein ◽  
Rnd Transporter ◽  
Lipid Environment

AbstractThe small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases the resistance of the bacterium to a subset of the antibiotic substrates of that transporter. It is not clear how the physical association of the two proteins selectively changes activity of the pump for defined substrates. Here, we report cryo-EM structures of AcrB and the AcrBZ complex in lipid environments, and comparisons suggest that conformational changes occur in the drug binding pocket as a result of AcrZ binding. Simulations indicate that cardiolipin preferentially interacts with the AcrBZ complex, due to increased contact surface, and we observe that the drug sensitivity of bacteria lacking AcrZ is exacerbated when combined with cardiolipin deficiency. Taken together, the data suggest that AcrZ and lipid cooperate to allosterically modulate the activity of AcrB. This mode of regulation by a small protein and lipid may occur for other membrane proteins.

scholarly journals Structural mechanism of transcription regulation of the Staphylococcus aureus multidrug efflux operon mepRA by the MarR family repressor MepR

2013 ◽  
Vol 42 (4) ◽  
pp. 2774-2788 ◽  
Author(s):  
Ivan Birukou ◽  
Susan M. Seo ◽  
Bryan D. Schindler ◽  
Glenn W. Kaatz ◽  
Richard G. Brennan
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Conformational Changes ◽  
Efflux Pump ◽  
Binding Activity ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Structural Mechanism ◽  
Dna Binding Activity ◽  
Complex Structural

Abstract The multidrug efflux pump MepA is a major contributor to multidrug resistance in Staphylococcus aureus. MepR, a member of the multiple antibiotic resistance regulator (MarR) family, represses mepA and its own gene. Here, we report the structure of a MepR–mepR operator complex. Structural comparison of DNA-bound MepR with ‘induced’ apoMepR reveals the large conformational changes needed to allow the DNA-binding winged helix-turn-helix motifs to interact with the consecutive major and minor grooves of the GTTAG signature sequence. Intriguingly, MepR makes no hydrogen bonds to major groove nucleobases. Rather, recognition-helix residues Thr60, Gly61, Pro62 and Thr63 make sequence-specifying van der Waals contacts with the TTAG bases. Removing these contacts dramatically affects MepR–DNA binding activity. The wings insert into the flanking minor grooves, whereby residue Arg87, buttressed by Asp85, interacts with the O2 of T4 and O4′ ribosyl oxygens of A23 and T4. Mutating Asp85 and Arg87, both conserved throughout the MarR family, markedly affects MepR repressor activity. The His14′:Arg59 and Arg10′:His35:Phe108 interaction networks stabilize the DNA-binding conformation of MepR thereby contributing significantly to its high affinity binding. A structure-guided model of the MepR–mepA operator complex suggests that MepR dimers do not interact directly and cooperative binding is likely achieved by DNA-mediated allosteric effects.

scholarly journals Structural and functional aspects of the multidrug efflux pump AcrB

2009 ◽  
Vol 390 (8) ◽  
Author(s):  
Thomas Eicher ◽  
Lorenz Brandstätter ◽  
Klaas M. Pos
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Efflux Pump ◽  
Binding Pocket ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Substrate Binding Pocket ◽  
Functional Aspects ◽  
Transport Cycle ◽  
Multiple Antibiotics

Abstract The tripartite efflux system AcrA/AcrB/TolC is the main pump in Escherichia coli for the efflux of multiple antibiotics, dyes, bile salts and detergents. The inner membrane component AcrB is central to substrate recognition and energy transduction and acts as a proton/drug antiporter. Recent structural studies show that homotrimeric AcrB can adopt different monomer conformations representing consecutive states in an allosteric functional rotation transport cycle. The conformational changes create an alternate access drug transport tunnel including a hydrophobic substrate binding pocket in one of the cycle intermediates.

Sign in / Sign up

Export Citation Format

Share Document