Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors.
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ABSTRACT:
SUBMITTER: Sennhauser G
PROVIDER: S-EPMC1717020 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H+/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.
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Project description:Multidrug resistance is a serious problem in current chemotherapy. The efflux system largely responsible for resistance in Escherichia coli contains the drug transporter, AcrB. The structures of AcrB were solved in 2002 as the symmetric homo-trimer, and then in 2006 as the asymmetric homo-trimer. The latter suggested a functionally rotating mechanism. Here, by molecular simulations of the AcrB porter domain, we uncovered allosteric coupling and the drug export mechanism in the AcrB trimer. Allosteric coupling stabilized the asymmetric structure with one drug molecule bound, which validated the modelling. Drug dissociation caused a conformational change and stabilized the symmetric structure, providing a unified view of the structures reported in 2002 and 2006. A dynamic study suggested that, among the three potential driving processes, only protonation of the drug-bound protomer can drive the functional rotation and simultaneously export the drug.
| S-EPMC3065909 | biostudies-literature
Project description:The overexpression of RND-type exporters is one of the main causes of multidrug resistance (MDR) in Gram-negative pathogens. In RND transporters, such as Escherichia coli's main efflux pump AcrB, drug efflux occurs in the porter domain, while protons flow through the transmembrane domain: remote conformational coupling. At the border of a transmembrane helix (TM8) and subdomain PC2, there is a loop which makes a hoisting movement by a random-coil-to-α-helix change, and opens and closes a drug channel entrance. This loop is supposed to play a key role in the allosteric conformational coupling between the transmembrane and porter domain. Here we show the results of a series of flexibility loop-mutants of AcrB. We determined the crystal structure of a three amino acid truncated loop mutant, which is still a functional transporter, and show that the short α-helix between Cβ15 and the loop unwinds to a random coil in the access and binding monomers and in the extrusion monomer it makes a partially stretched coil-to-helix change. The loop has undergone compensatory conformational changes and still facilitates the opening and closing of the channel. In addition, more flexible mutated loops (proline mutated and significantly elongated) can still function during export. The flexibility in this region is however limited, as an even more truncated mutant (six amino acid deletion) becomes mostly inactive. We found that the hoisting-loop is a highly flexible hinge that enables the conformational energy transmission passively.
| S-EPMC5661021 | biostudies-literature
Project description:Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action is poorly understood. Exposure of Salmonella enterica serovar Typhimurium and Escherichia coli to chlorpromazine selected for mutations within genes encoding RamR and MarR, regulators of the multidrug tripartite efflux pump AcrAB-TolC. Further experiments with S. Typhimurium containing AcrB D408A (a nonfunctional efflux pump) and chlorpromazine or amitriptyline resulted in the reversion of the mutant acrB allele to the wild type. Together, this suggests these drugs are AcrB efflux substrates. Subsequent docking studies with AcrB from S. Typhimurium and E. coli, followed by molecular dynamics simulations and free energy calculations showed that chlorpromazine and amitriptyline bind at the hydrophobic trap, a preferred binding site for substrates and inhibitors within the distal binding pocket of AcrB. Based on these simulations, we suggest that chlorpromazine and amitriptyline inhibit AcrB-mediated efflux by interfering with substrate binding. Our findings provide evidence that these drugs are substrates and inhibitors of AcrB, yielding molecular details of their mechanism of action and informing drug discovery of new efflux inhibitors.IMPORTANCE Efflux pumps of the resistance nodulation-cell division (RND) superfamily are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; however, several issues have thus far hindered all efforts at designing new efflux inhibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibitors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds.
| S-EPMC7267879 | biostudies-literature
Project description:AcrB is the inner membrane transporter of the tripartite multidrug efflux pump AcrAB-TolC in E. coli, which poses a major obstacle to the treatment of bacterial infections. X-ray structures have identified two types of substrate-binding pockets in the porter domains of AcrB trimer: the proximal binding pocket (PBP) and the distal binding pocket (DBP), and suggest a functional rotating mechanism in which each protomer cycles consecutively through three distinct conformational states (access, binding and extrusion). However, the details of substrate binding and translocation between the binding pockets remain elusive. In this work, we performed atomic simulations to obtain the free energy profile of the translocation of an antibiotic drug doxorubicin (DOX) inside AcrB. Our simulation indicates that DOX binds at the PBP and DBP with comparable affinities in the binding state protomer, and overcomes a 3 kcal/mol energy barrier to transit between them. Obvious conformational changes including closing of the PC1/PC2 cleft and shrinking of the DBP were observed upon DOX binding in the PBP, resulting in an intermediate state between the access and binding states. Taken together, the simulation results reveal a detailed stepwise substrate binding and translocation process in the framework of functional rotating mechanism.
| S-EPMC4595977 | biostudies-literature
Project description:Multidrug ATP-Binding Cassette (ABC) exporters use an alternating access mechanism to translocate drugs across membranes, but the molecular mechanism of substrate release is still elusive due to the poor affinity of the drug-binding pocket for its ligand at this stage. Here, we bring new information on this mechanism by resolving two ATP-bound outward-facing conformations of the homodimer BmrA from Bacillus subtilis by X-ray crystallography with no drug bound and by cryo-EM in the presence of rhodamine 6G. This revealed the first structure of a multidrug pump with a substrate bound to the drug-binding pocket in a pre-release state. Two molecules of the dye are located in an hydrophobic region at the level of the outer leaflet between transmembrane (TM) helices 1, 2 and 6. Markedly, the X-ray structures display a half-closed TM1-2 region that is widely open in the cryo-EM one. Hydrogen-Deuterium exchange coupled to mass spectrometry (HDX-MS) and molecular dynamics simulations confirmed the high flexibility of this region, presumably driving the release of structurally divergent compounds.
2021-03-25 | PXD022185 | Pride
Project description:Many proteins, especially transporters, are thought to undergo large conformational alterations during their functional cycle. Since X-ray crystallography usually gives only the most stable conformation, other methods are needed to probe this conformational change. Site-directed disulfide cross-linking is often very useful for this purpose. We illustrate this by using the Escherichia coli AcrB, a proton-motive-force-dependent multidrug efflux transporter. 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). Site-directed disulfide cross-linking between those residues that come close to each other in only one stage in the cycle inactivated the transporter, showing that the conformational changes indeed occurred in vivo and that they are required for drug transport. A dsbA strain, which has a diminished activity to form disulfide bonds in the periplasm, was used to verify the conclusion by showing a restored transport activity in this strain. Furthermore, we describe "a real-time cross-linking experiment," in which rapidly reacting, sulfhydryl-specific cross-linkers, methanethiosulfonates, inactivate the AcrB double-cysteine mutant expressed in dsbA cells instantaneously.
| S-EPMC2915569 | biostudies-literature
Project description: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.
| S-EPMC1636240 | biostudies-literature
Project description:CmABCB1 is a Cyanidioschyzon merolae homolog of human ABCB1, a well known ATP-binding cassette (ABC) transporter responsible for multi-drug resistance in various cancers. Three-dimensional structures of ABCB1 homologs have revealed the snapshots of inward- and outward-facing states of the transporters in action. However, sufficient information to establish the sequential movements of the open-close cycles of the alternating-access model is still lacking. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has proven its worth in determining novel structures and recording sequential conformational changes of proteins at room temperature, especially for medically important membrane proteins, but it has never been applied to ABC transporters. In this study, 7.7 mono-acyl-glycerol with cholesterol as the host lipid was used and obtained well diffracting microcrystals of the 130 kDa CmABCB1 dimer. Successful SFX experiments were performed by adjusting the viscosity of the crystal suspension of the sponge phase with hy-droxy-propyl methyl-cellulose and using the high-viscosity sample injector for data collection at the SACLA beamline. An outward-facing structure of CmABCB1 at a maximum resolution of 2.22 Å is reported, determined by SFX experiments with crystals formed in the lipidic cubic phase (LCP-SFX), which has never been applied to ABC transporters. In the type I crystal, CmABCB1 dimers interact with adjacent molecules via not only the nucleotide-binding domains but also the transmembrane domains (TMDs); such an interaction was not observed in the previous type II crystal. Although most parts of the structure are similar to those in the previous type II structure, the substrate-exit region of the TMD adopts a different configuration in the type I structure. This difference between the two types of structures reflects the flexibility of the substrate-exit region of CmABCB1, which might be essential for the smooth release of various substrates from the transporter.
| S-EPMC8733880 | biostudies-literature
Project description:Efflux pumps of the resistance nodulation division (RND) superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. The development of inhibitors of the RND pumps would improve the efficacy of current and next-generation antibiotics. To date, however, only one inhibitor has been cocrystallized with AcrB. Thus, in silico structure-based analysis is essential for elucidating the interaction between other inhibitors and the efflux pumps. In this work, we used computer docking and molecular dynamics simulations to study the interaction between AcrB and the compound MBX2319, a novel pyranopyridine efflux pump inhibitor with potent activity against RND efflux pumps of Enterobacteriaceae species, as well as other known inhibitors (D13-9001, 1-[1-naphthylmethyl]-piperazine, and phenylalanylarginine-β-naphthylamide) and the binding of doxorubicin to the efflux-defective F610A variant of AcrB. We also analyzed the binding of a substrate, minocycline, for comparison. Our results show that MBX2319 binds very tightly to the lower part of the distal pocket in the B protomer of AcrB, strongly interacting with the phenylalanines lining the hydrophobic trap, where the hydrophobic portion of D13-9001 was found to bind by X-ray crystallography. Additionally, MBX2319 binds to AcrB in a manner that is similar to the way in which doxorubicin binds to the F610A variant of AcrB. In contrast, 1-(1-naphthylmethyl)-piperazine and phenylalanylarginine-β-naphthylamide appear to bind to somewhat different areas of the distal pocket in the B protomer of AcrB than does MBX2319. However, all inhibitors (except D13-9001) appear to distort the structure of the distal pocket, impairing the proper binding of substrates.
| S-EPMC4187987 | biostudies-literature