Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB

Eicher, Thomas; Seeger, Markus A.; Anselmi, Claudio; Zhou, Wenchang; Brandstätter, Lorenz; Verrey, François; Diederichs, Kay; Faraldo‐Gómez, José D.; Pos, Klaas M.

doi:10.7554/elife.03145

Cited by 156 publications

(351 citation statements)

References 52 publications

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“…The V127A/G and D153E substitutions appear to influence events at the periphery or the hypothetical extended drug binding pocket, while the Y49S substitution may affect steps leading to the drug exit path. Curiously, two alterations mapped to the transmembrane helices 5 and 6 (TM5 and TM6), which are in the proximity of TM4, which houses the D407 and D408 residues that are critical for the proton relay network and thus the pump's drug translocation activity in the periplasmic domain (25). Despite affecting two different domains of AcrB, all seven alterations were found in the first half of the protein, delineated by TM1 to TM6 and the periplasmic domain protruding from TM1 and TM2.…”

Section: Resultsmentioning

confidence: 99%

“…The TM domain houses five residues-D407, D408, K940, R971, and T978 -which are critical for proton translocation and AcrB activity (19,(22)(23)(24)(25). The movement of protons through these residues is coupled to the conformational changes in the periplasmic domain that are essential for drug binding and extrusion.…”

Section: Discussionmentioning

confidence: 99%

“…The movement of protons through these residues is coupled to the conformational changes in the periplasmic domain that are essential for drug binding and extrusion. Although F453 and L486 are not directly connected to the proton relay network, it has been proposed that D408 transiently interacts through the backbone of L442 (TM5) and hydrogen bonds with S481 (TM6) (25). Therefore, the F453C and L486W substitutions could potentially influence these interactions or establish novel interactions within the TM region that indirectly, albeit positively, influence events at the periplasmic domain, including transitioning of the protomers to three distinct conformational states.…”

Section: Discussionmentioning

confidence: 99%

“…The TM domain contains five highly conserved residues-D407, D408, K940, R971, and T978 -of the proton relay network that are critical for AcrB function (19,(22)(23)(24)(25). Based on different functional implications, the periplasmic domain has been divided into a membrane-proximal porter domain and a membrane-distal docking domain (4).…”

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confidence: 99%

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

et al. 2015

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The AcrB protein of Escherichia 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 of N-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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

et al. 2015

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“…The AcrAB-TolC system belongs to the RND (resistance-nodulation-cell division)-type transporter family and is composed of an inner membrane transporter (AcrB), an outer membrane channel (TolC), and an adaptor protein (AcrA) (10)(11)(12). The crystal structures of each component were elucidated in the early 2000s (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The AcrA molecule can be completely replaced by the highly homologous adaptor AcrE (25), which is induced under acrABdeficient conditions (26)(27)(28)(29).…”

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confidence: 99%

AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

et al. 2016

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The AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB-deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function. IMPORTANCEThe structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB-deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for considering the structure and mechanism of AcrABTolC-mediated multidrug export.

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Bacterial Efflux Pumps and Their Inhibitors

Zgurskaya

Rybenkov

Walker

et al. 2021

Burger's Medicinal Chemistry and Drug Discovery

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Bacterial multidrug efflux transporters have distinct molecular features and mechanisms that enable their impressive substrate polyspecificity. These transporters are involved in all physiological processes where nonspecific but active transport across membranes is critical for survival and proliferation. Importantly, efflux pumps contribute to bacterial virulence and pathogenesis, as well as the spread of antibiotic resistance in clinics. Transporters belonging to several protein superfamilies have been selected for polyspecificity by evolution and augmented with accessory proteins that extend and couple transport reactions across the inner and outer membranes of the bacterium. As a result, bacteria are equipped with diverse efflux machines that protect cells from a broad variety of toxic compounds including antibiotics and other small molecule therapeutics. The inhibition of such polyspecific biomolecular machines is a challenging task, which still awaits an efficient solution. Here, we review the molecular mechanisms of efflux pumps from Gram‐negative bacteria and recent advances and challenges associated with the discovery of effective inhibitors of these pumps.

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Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB

Cited by 156 publications

References 52 publications

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

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

AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

Bacterial Efflux Pumps and Their Inhibitors

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