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 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.

scholarly journals Characterization and Molecular Determinants for β-Lactam Specificity of the Multidrug Efflux Pump AcrD from Salmonella typhimurium

Antibiotics ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1494
Author(s):  
Jenifer Cuesta Bernal ◽  
Jasmin El-Delik ◽  
Stephan Göttig ◽  
Klaas M. Pos
Keyword(s):  
Substrate Specificity ◽  
Salmonella Typhimurium ◽  
Efflux Pump ◽  
Binding Pocket ◽  
Salt Resistance ◽  
Drug Binding ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Molecular Determinants ◽  
Groove Region

Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.

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 Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

2015 ◽  
Vol 197 (20) ◽  
pp. 3255-3264 ◽  
Author(s):  
Ketaki Soparkar ◽  
Alfred D. Kinana ◽  
Jon W. Weeks ◽  
Keith D. Morrison ◽  
Hiroshi Nikaido ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Conformational Dynamics ◽  
Binding Pocket ◽  
Drug Binding ◽  
Single Amino Acid ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Isolation And Characterization ◽  
Efflux Kinetics

ABSTRACTThe AcrB protein ofEscherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions—Y49S, V127A, V127G, D153E, and G288C—mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions—F453C and L486W—were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux ofN-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain.IMPORTANCEThe AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.

scholarly journals The assembled structure of a complete tripartite bacterial multidrug efflux pump

2009 ◽  
Vol 106 (17) ◽  
pp. 7173-7178 ◽  
Author(s):  
Martyn F. Symmons ◽  
Evert Bokma ◽  
Eva Koronakis ◽  
Colin Hughes ◽  
Vassilis Koronakis
Keyword(s):  
Efflux Pump ◽  
Cell Envelope ◽  
Adaptor Protein ◽  
Drug Binding ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Inner Membrane ◽  
Multidrug Efflux Pumps ◽  
Docking Approach

Bacteria likeEscherichia coliandPseudomonas aeruginosaexpel drugs via tripartite multidrug efflux pumps spanning both inner and outer membranes and the intervening periplasm. In these pumps a periplasmic adaptor protein connects a substrate-binding inner membrane transporter to an outer membrane-anchored TolC-type exit duct. High-resolution structures of all 3 components are available, but a pump model has been precluded by the incomplete adaptor structure, because of the apparent disorder of its N and C termini. We reveal that the adaptor termini assemble a β-roll structure forming the final domain adjacent to the inner membrane. The completed structure enabled in vivo cross-linking to map intermolecular contacts between the adaptor AcrA and the transporter AcrB, defining a periplasmic interface between several transporter subdomains and the contiguous β-roll, β-barrel, and lipoyl domains of the adaptor. With short and long cross-links expressed as distance restraints, the flexible linear topology of the adaptor allowed a multidomain docking approach to model the transporter–adaptor complex, revealing that the adaptor docks to a transporter region of comparative stability distinct from those key to the proposed rotatory pump mechanism, putative drug-binding pockets, and the binding site of inhibitory DARPins. Finally, we combined this docking with our previous resolution of the AcrA hairpin–TolC interaction to develop a model of the assembled tripartite complex, satisfying all of the experimentally-derived distance constraints. This AcrA3-AcrB3-TolC3model presents a 610,000-Da, 270-Å-long efflux pump crossing the entire bacterial cell envelope.

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.

scholarly journals Interchangeability of Periplasmic Adaptor Proteins AcrA and AcrE in forming functional efflux pumps with AcrD in Salmonella Typhimurium

2021 ◽  
Author(s):  
Ilyas Alav ◽  
Vassiliy N Bavro ◽  
Jessica M A Blair
Keyword(s):  
Drug Targets ◽  
Efflux Pump ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Interaction Sites ◽  
Functional Complex ◽  
Rnd Transporter

Background RND efflux pumps are important mediators of antibiotic resistance. RND pumps including the principal multidrug-efflux pump AcrAB-TolC in Salmonella, are tripartite systems, with an inner membrane RND-transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor (OMF). We previously identified the residues required for binding between the PAP AcrA and the RND-transporter AcrB and have demonstrated that PAPs can function with non-cognate transporters. AcrE and AcrD/AcrF are homologues of AcrA and AcrB, respectively. Here, we show that AcrE can interact with AcrD, which does not possess its own PAP, and establish that the residues previously identified in AcrB-binding are also involved in AcrD-binding. Methods The acrD and acrE genes were expressed into a strain lacking acrABDEF (Δ3RND). PAP residues involved in promiscuous interactions were predicted based on previously defined PAP-RND interactions and corresponding mutations generated in acrA and acrE. Antimicrobial susceptibility of the mutant strains was determined. Results Co-expression of acrD and acrE significantly decreased susceptibility of the Δ3RND strain to AcrD substrates showing that AcrE can form a functional complex with AcrD. The substrate profile of Salmonella AcrD differed from that of E. coli AcrD. Mutations targeting the previously defined PAP-RND interaction sites in AcrA/AcrE impaired efflux of AcrD-dependent substrates. Conclusions These data indicate that AcrE forms an efflux-competent pump with AcrD and thus presents an alternative PAP for this pump. Mutagenesis of the conserved RND binding sites validates the interchangeability of AcrA and AcrE, highlighting them as potential drug targets for efflux inhibition.

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