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HDX-MS reveals the perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

ABSTRACT: Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

INSTRUMENT(S): Synapt MS

ORGANISM(S): Escherichia Coli

SUBMITTER: Zainab Ahdash

LAB HEAD: Argyris Politis

PROVIDER: PXD019047 | Pride | 2020-10-07

REPOSITORIES: Pride

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			Action	DRS
	190318_AcrB_G288D_ref_1_IA_final_peptide.csv	Csv
	190318_AcrB_G288D_ref_3_IA_final_peptide.csv	Csv
	190318_AcrB_G288D_ref_5_IA_final_peptide.csv	Csv
	190318_AcrB_wt_ref_1_IA_final_peptide.csv	Csv
	190318_AcrB_wt_ref_3_IA_final_peptide.csv	Csv

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Publications

Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB.

Reading Eamonn E Ahdash Zainab Z Fais Chiara C Ricci Vito V Wang-Kan Xuan X Grimsey Elizabeth E Stone Jack J Malloci Giuliano G Lau Andy M AM Findlay Heather H Konijnenberg Albert A Booth Paula J PJ Ruggerone Paolo P Vargiu Attilio V AV Piddock Laura J V LJV Politis Argyris A

Nature communications 20201104 1

Resistance-nodulation-division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry ...[more]

PMID: 33149158

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Mutations in Multidrug Efflux Pumps of Eshcerichia coli

Project description:The number and overlapping substrate repertoire of multidrug efflux pumps in the E. coli genome suggest a physiological role apart from multidrug resistance. This role was investigated using transcriptomic analyses of cDNAs labeled from E. coli AG102 mRNA (hyper drug resistant, marR1) and its isogenic major efflux pump mutants. Keywords: Mutation Analysis

2008-03-11 | GSE10757 | GEO

Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB.

Project description:Resistance-nodulation-division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics.

| S-EPMC7642415 | biostudies-literature

Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein

Project description:Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αβ domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.

2023-06-01 | PXD041359 | Pride

Structural insights into the catalytic cycle of a bacterial multidrug ABC efflux pump

Project description:ABC (“ATP-Binding Cassette”) exporters of the type IV subfamily gather transporters involved in the efflux of many compounds and notably those capable to confer multidrug resistance like the mammalian P-glycoprotein or the bacterial transporter BmrA. They function according to an alternating access mechanism between inward-facing (IF) and outward-facing (OF) conformations, but the extent of physical separation between the two nucleotide-binding domains (NBDs) in different states is still unsettled. Small Angle Neutron Scattering (SANS) and hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) were used to highlight different conformational states of BmrA during its catalytic cycle. In particular, mutation of the invariant Lysine residue of the Walker-A motif (K380A) captures BmrA in an ATP-bound IF conformation prior to NBD closure. While in the transition-like state induced by vanadate, wild-type BmrA is mainly in an OF conformation, it and populates only IF conformations only in the presence of ADP/Mg. Importantly, in this post-hydrolytic step, distances between the two NBDs of BmrA appears to be more separated than in the apo state, but they remains shorter than in the widest opening found in the related MsbA transporter. Overall, our results highlight the main steps of the catalytic cycle of a homodimeric bacterial multidrug transporter and underline structural and functional commonalities as well as oddities among the type IV subfamily of ABC exporters.

2022-03-24 | PXD028663 | Pride

Real-time visualization of drug resistance acquisition by horizontal gene transfer reveals a new role for AcrAB-TolC multidrug efflux pump

Project description:In our study, we obtained evidence showing that, in the presence of tetracycline, AcrAB-TolC multidrug efflux pump was required for production of protein encoded by a conjugative plasmid acquired by bacterial conjugation. We then address the role of AcrAB-TolC multidrug efflux pump on the level of protein synthesis activity in the presence of tetracycline, which inhibits protein translation by targeting ribosomal 16S rRNA and competing with binding of the anticodon-stem loop of an A-site tRNA. We used a quantitative TMT-nanoLC-mass spectrometry approach to obtained the protein relative abundance ratio before and after incubation with tetracycline, and so in wild-type(MG1655, K12) and AcrAB-TolC mutant strains (ΔacrA, ΔacrB and ΔtolC).

2019-07-16 | PXD011286 | Pride

HDX-MS studies of the bacterial multidrug ABC transporter BmrA in presence or absence of a ligand (rhodamine-6G)

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 R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5’-6’ of the other. 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.

2022-01-27 | PXD027447 | Pride

Hfq Regulates Efflux Pump Expression and Purine Metabolic Pathway to Increase the Trimethoprim Resistance in Aeromonas veronii

Project description:<p>Aeromonas veronii (A. veronii) is a zoonotic pathogen. It causes clinically a variety of diseases such as dysentery, bacteremia, and meningitis. And it also brings huge losses to aquaculture. A. veronii has been documented as a multiple antibiotic resistant bacterium. Hfq (host factor for RNA bacteriophage Qβ replication) plays vital regulation roles in a variety of cell programs. The deletion of hfq in A. veronii showed an increased sensitivity to the trimethoprim, accompanying by the down regulation of the efflux pump related genes acrA and acrB in transcriptomic data. Coherently, the complementation of acrA and acrB in ∆hfq recovered the trimethoprim resistance. Besides, the productions of adenosine and guanosine were revealed to augment in ∆hfq in metabonomic data. Both wild type and ∆hfq conferred more sensitive to trimethoprim after appending 1 mM adenosine or guanosine to M9 medium. These results demonstrated that Hfq mediated trimethoprim resistance by elevating efflux pump expression and degrading adenosine, and guanosine metabolites. Collectively, Hfq is a potential target to tackle trimethoprim resistance in A. veronii infection.</p>

2020-02-19 | MTBLS1411 | MetaboLights

The impaired quorum sensing response of MexAB-OprM efflux pump overexpressing mutants is not due to non-physiological efflux of 3-oxo-C12-HSL

Project description:Multidrug (MDR) efflux pumps are ancient and conserved molecular machineries with relevant roles in different aspects of the bacterial physiology, besides antibiotic resistance. In the case of the environmental opportunistic pathogen Pseudomonas aeruginosa, it has been shown that overexpression of different efflux pumps is linked to the impairment of the quorum sensing (QS) response. Nevertheless, the causes of such impairment are different for each analyzed efflux pump. Herein, we performed an in-depth analysis of the QS-mediated response of a P. aeruginosa antibiotic resistant mutant that overexpresses MexAB-OprM. Although previous work claimed that this efflux pump extrudes the QS signal 3-oxo-C12-HSL, we show otherwise. Our results evidence that the observed attenuation in the QS response when overexpressing this pump is related to an impaired production of alkyl quinolone QS signals, likely prompted by the reduced availability of one of their precursors, the octanoate. Together with previous studies, this indicates that, although the consequences of overexpressing efflux pumps are similar (impaired QS response), the underlying mechanisms are different. This ‘apparent redundancy' of MDR efflux systems can be understood as a P. aeruginosa strategy to keep the robustness of the QS regulatory network and modulate its output in response to different signals.

2021-10-25 | GSE153996 | GEO

Outward-facing structures of a multidrug ABC exporter with and without bound drug

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

A novel TetR-like transcriptional regulator is induced in acid-nitrosative stress and controls expression of an efflux pump in mycobacteria

Project description:Mycobacterium tuberculosis has the ability to survive inside macrophages under acid-nitrosative stress. M. tuberculosis Rv1685c and its orthologue in M. smegmatis, MSMEG_3765, are induced on exposure to acid-nitrosative stress. Both genes are annotated as TetR transcriptional regulators, a family of proteins that regulate a wide range of cellular activities, including multidrug resistance, carbon catabolism and virulence. Here we demonstrate that MSMEG_3765 is co-transcribed with the upstream genes MSMEG_3762 and MSMEG_3763, encoding efflux pump components. RTq-PCR and GFP-reporter assays showed that the MSMEG_3762/63/65 gene cluster, and the orthologous region in M. tuberculosis (Rv1687c/86c/85c), was up-regulated in a MSMEG_3765 null mutant, suggesting that MSMEG_3765 acts as a repressor, typical of this family of regulators. We further defined the MSMEG_3765 regulon using genome-wide transcriptional profiling and confirmed that the MSMEG_3762/63/65 promoter is induced under acid-nitrosative stress. A putative 36 bp regulatory motif was identified upstream of the gene clusters in both M. smegmatis and M. tuberculosis and purified recombinant MSMEG_3765 protein was found to bind to DNA fragments containing this motif from both M. smegmatis and M. tuberculosis upstream regulatory regions. These results suggest that the TetR repressor MSMEG_3765/Rv1685c controls expression of an efflux pump with an, as yet, undefined role in the mycobacterial response to acid-nitrosative stress.

2017-10-24 | E-MTAB-5869 | biostudies-arrayexpress