scholarly journals A Model for Allosteric Communication in Drug Transport by the AcrAB-TolC Tripartite Efflux Pump

Antibiotics ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 52
Author(s):  
Anya Webber ◽  
Malitha Ratnaweera ◽  
Andrzej Harris ◽  
Ben F. Luisi ◽  
Véronique Yvette Ntsogo Enguéné
Keyword(s):  
Membrane Protein ◽  
Conformational Changes ◽  
Drug Transport ◽  
Considerable Increase ◽  
Efflux Pump ◽  
Transition Process ◽  
Membrane Fusion Protein ◽  
Allosteric Communication ◽  
Inner Membrane Protein ◽  
Dynamics Simulations

RND family efflux pumps are complex macromolecular machines involved in multidrug resistance by extruding antibiotics from the cell. While structural studies and molecular dynamics simulations have provided insights into the architecture and conformational states of the pumps, the path followed by conformational changes from the inner membrane protein (IMP) to the periplasmic membrane fusion protein (MFP) and to the outer membrane protein (OMP) in tripartite efflux assemblies is not fully understood. Here, we investigated AcrAB-TolC efflux pump’s allostery by comparing resting and transport states using difference distance matrices supplemented with evolutionary couplings data and buried surface area measurements. Our analysis indicated that substrate binding by the IMP triggers quaternary level conformational changes in the MFP, which induce OMP to switch from the closed state to the open state, accompanied by a considerable increase in the interface area between the MFP subunits and between the OMPs and MFPs. This suggests that the pump’s transport-ready state is at a more favourable energy level than the resting state, but raises the puzzle of how the pump does not become stably trapped in a transport-intermediate state. We propose a model for pump allostery that includes a downhill energetic transition process from a proposed ‘activated’ transport state back to the resting pump.

scholarly journals BpeAB-OprB, a Multidrug Efflux Pump in Burkholderia pseudomallei

2004 ◽  
Vol 48 (4) ◽  
pp. 1128-1135 ◽  
Author(s):  
Y. Y. Chan ◽  
T. M. C. Tan ◽  
Y. M. Ong ◽  
K. L. Chua
Keyword(s):  
Membrane Protein ◽  
Burkholderia Pseudomallei ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Membrane Fusion Protein ◽  
Wide Range ◽  
Inner Membrane Protein

ABSTRACT Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents, including β-lactams, aminoglycosides, macrolides, and polymyxins. An operon, bpeR-bpeA-bpeB-oprB, which encodes a putative repressor, a membrane fusion protein, an inner membrane protein, and an outer membrane protein, respectively, of a multidrug efflux pump of the resistance-nodulation-division family was identified in B. pseudomallei. The divergently transcribed bpeR gene encodes a putative repressor protein of the TetR family which probably regulates the expression of the bpeAB-oprB gene cluster. Comparison of the MICs and minimal bactericidal concentrations of antimicrobials for bpeAB deletion mutant KHWΔbpeAB and its isogenic wild-type parent, KHW, showed that the B. pseudomallei BpeAB-OprB pump is responsible for the efflux of the aminoglycosides gentamicin and streptomycin, the macrolide erythromycin, and the dye acriflavine. Antibiotic efflux by the BpeAB-OprB pump was dependent on a proton gradient and differs from that by the AmrAB-OprA pump in that it did not efflux the aminoglycoside spectinomycin or the macrolide clarithromycin. The broad-spectrum efflux pump inhibitor MC-207,110 did not potentiate the effectiveness of the antimicrobials erythromycin and streptomycin in B. pseudomallei.

scholarly journals The MexJK Efflux Pump of Pseudomonas aeruginosa Requires OprM for Antibiotic Efflux but Not for Efflux of Triclosan

2002 ◽  
Vol 184 (18) ◽  
pp. 5036-5044 ◽  
Author(s):  
Rungtip Chuanchuen ◽  
Craig T. Narasaki ◽  
Herbert P. Schweizer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Efflux Pump ◽  
Resistant Mutant ◽  
Efflux System ◽  
Membrane Fusion Protein ◽  
Single Nucleotide Change ◽  
Antibiotic Efflux

ABSTRACT Using the biocide triclosan as a selective agent, several triclosan-resistant mutants of a susceptible Pseudomonas aeruginosa strain were isolated. Cloning and characterization of a DNA fragment conferring triclosan resistance from one of these mutants revealed a hitherto uncharacterized efflux system of the resistance nodulation cell division (RND) family, which was named MexJK and which is encoded by the mexJK operon. Expression of this operon is negatively regulated by the product of mexL, a gene located upstream of and transcribed divergently from mexJK. The triclosan-resistant mutant contained a single nucleotide change in mexL, which caused an amino acid change in the putative helix-turn-helix domain of MexL. The MexL protein belongs to the TetR family of repressor proteins. The MexJK system effluxed tetracycline and erythromycin but only in the presence of the outer membrane protein channel OprM; OprJ and OprN did not function with MexJK. Triclosan efflux required neither of the outer membrane protein channels tested but necessitated the MexJ membrane fusion protein and the MexK inner membrane RND transporter. The results presented in this study suggest that MexJK may function as a two-component RND pump for triclosan efflux but must associate with OprM to form a tripartite antibiotic efflux system. Furthermore, the results confirm that triclosan is an excellent tool for the study of RND multidrug efflux systems and that this popular biocide therefore readily selects mutants which are cross-resistant with antibiotics.

scholarly journals Quantum dot probes for the quantitative study of drug transport by the MacAB TolC efflux pump in lipid scaffolds

2020 ◽  
Author(s):  
Hager Souabni ◽  
William Batista dos Santos ◽  
Quentin Cece ◽  
Dhenesh Puvanendran ◽  
Martin Picard
Keyword(s):  
Energy Consumption ◽  
Membrane Protein ◽  
Virulence Factors ◽  
Quantitative Study ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Efflux Pump ◽  
Coupling Efficiency ◽  
Gram Negative Bacteria ◽  
Gram Negative

AbstractABC tripartite efflux pumps are macromolecular membrane protein machineries that expel a large variety of drugs and export virulence factors from Gram negative bacteria. Using a lipid scaffold mimicking the two-membrane environment of the transporter and designing spectroscopic conditions allowing the monitoring of both ATP hydrolysis and substrate transport in real time, we show that MacAB-TolC accommodates transport and energy consumption with high coupling efficiency.

scholarly journals Insights into the Role of Membrane Lipids in the Structure, Function and Regulation of Integral Membrane Proteins

2021 ◽  
Vol 22 (16) ◽  
pp. 9026
Author(s):  
Kenta Renard ◽  
Bernadette Byrne
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Membrane Lipids ◽  
Protein Protein Interactions ◽  
Technological Advances ◽  
Highly Hydrophobic ◽  
Dynamics Simulations ◽  
And Function

Membrane proteins exist within the highly hydrophobic membranes surrounding cells and organelles, playing key roles in cellular function. It is becoming increasingly clear that the membrane does not just act as an appropriate environment for these proteins, but that the lipids that make up these membranes are essential for membrane protein structure and function. Recent technological advances in cryogenic electron microscopy and in advanced mass spectrometry methods, as well as the development of alternative membrane mimetic systems, have allowed experimental study of membrane protein–lipid complexes. These have been complemented by computational approaches, exploiting the ability of Molecular Dynamics simulations to allow exploration of membrane protein conformational changes in membranes with a defined lipid content. These studies have revealed the importance of lipids in stabilising the oligomeric forms of membrane proteins, mediating protein–protein interactions, maintaining a specific conformational state of a membrane protein and activity. Here we review some of the key recent advances in the field of membrane protein–lipid studies, with major emphasis on respiratory complexes, transporters, channels and G-protein coupled receptors.

scholarly journals Computational Insights into Allosteric Conformational Modulation of P-Glycoprotein by Substrate and Inhibitor Binding

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 6006
Author(s):  
Juan Xing ◽  
Shuheng Huang ◽  
Yu Heng ◽  
Hu Mei ◽  
Xianchao Pan
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Molecular Design ◽  
Conformational Dynamics ◽  
Water Environment ◽  
Inhibitor Binding ◽  
Binding Domains ◽  
Allosteric Communication ◽  
P Glycoprotein ◽  
Dynamics Simulations

The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) is a physiologically essential membrane protein that protects many tissues against xenobiotic molecules, but limits the access of chemotherapeutics into tumor cells, thus contributing to multidrug resistance. The atomic-level mechanism of how substrates and inhibitors differentially affect the ATP hydrolysis by P-gp remains to be elucidated. In this work, atomistic molecular dynamics simulations in an explicit membrane/water environment were performed to explore the effects of substrate and inhibitor binding on the conformational dynamics of P-gp. Distinct differences in conformational changes that mainly occurred in the nucleotide-binding domains (NBDs) were observed from the substrate- and inhibitor-bound simulations. The binding of rhodamine-123 can increase the probability of the formation of an intermediate conformation, in which the NBDs were closer and better aligned, suggesting that substrate binding may prime the transporter for ATP hydrolysis. By contrast, the inhibitor QZ-Leu stabilized NBDs in a much more separated and misaligned conformation, which may result in the deficiency of ATP hydrolysis. The significant differences in conformational modulation of P-gp by substrate and inhibitor binding provided a molecular explanation of how these small molecules exert opposite effects on the ATPase activity. A further structural analysis suggested that the allosteric communication between transmembrane domains (TMDs) and NBDs was primarily mediated by two intracellular coupling helices. Our computational simulations provide not only valuable insights into the transport mechanism of P-gp substrates, but also for the molecular design of P-gp inhibitors.

scholarly journals Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

2019 ◽  
Author(s):  
Antonio N. Calabrese ◽  
Bob Schiffrin ◽  
Matthew Watson ◽  
Theodoros K. Karamanos ◽  
Martin Walko ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Single Molecule ◽  
Conformational Changes ◽  
Outer Membrane Protein ◽  
Conformational Dynamics ◽  
Core Domain ◽  
The Core ◽  
Single Molecule Fret ◽  
Dynamics Simulations

AbstractThe periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but how it binds its OMP clients and the mechanism(s) of its chaperone action remain unclear. Here, we have used chemical cross-linking, hydrogen-deuterium exchange, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and to interrogate the role of conformational dynamics of the chaperone’s domains in OMP recognition. We demonstrate that SurA samples a broad array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested by its crystal structure. Multiple binding sites for OMPs are located primarily in the core domain, with binding of the unfolded OMP resulting in conformational changes between the core/P1 domains. Together, the results portray a model in which unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between its domains assisting OMP recognition, binding and release.

scholarly journals Computational Study of Correlated Domain Motions in the AcrB Efflux Transporter

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Robert Schulz ◽  
Attilio V. Vargiu ◽  
Paolo Ruggerone ◽  
Ulrich Kleinekathöfer
Keyword(s):  
Transmembrane Domain ◽  
Efflux Pump ◽  
Computational Study ◽  
Mechanical Energy ◽  
Cooperative Effects ◽  
E Coli ◽  
Membrane Fusion Protein ◽  
Correlated Motions ◽  
Dynamics Simulations ◽  
Domain Motions

As active part of the major efflux system inE. colibacteria, AcrB is responsible for the uptake and pumping of toxic substrates from the periplasm toward the extracellular space. In combination with the channel protein TolC and membrane fusion protein AcrA, this efflux pump is able to help the bacterium to survive different kinds of noxious compounds. With the present study we intend to enhance the understanding of the interactions between the domains and monomers, for example, the transduction of mechanical energy from the transmembrane domain into the porter domain, correlated motions of different subdomains within monomers, and cooperative effects between monomers. To this end, targeted molecular dynamics simulations have been employed either steering the whole protein complex or specific parts thereof. By forcing only parts of the complex towards specific conformational states, the risk for transient artificial conformations during the simulations is reduced. Distinct cooperative effects between the monomers in AcrB have been observed. Possible allosteric couplings have been identified providing microscopic insights that might be exploited to design more efficient inhibitors of efflux systems.

scholarly journals Dynamic Community Composition Unravels Allosteric Communication in PDZ3

2020 ◽  
Author(s):  
Tandac F. Guclu ◽  
Ali Rana Atilgan ◽  
Canan Atilgan
Keyword(s):  
Community Composition ◽  
Conformational Changes ◽  
Structural Changes ◽  
Distal Segment ◽  
Allosteric Communication ◽  
C Terminus ◽  
N Terminus ◽  
Ligand Interactions ◽  
Binding Cavity ◽  
Dynamics Simulations

ABSTRACTThe third domain of PSD-95 (PDZ3) is a model for investigating allosteric communication in protein and ligand interactions. While motifs contributing to its binding specificity have been scrutinized, a conformational dynamical basis is yet to be established. Despite the miniscule structural changes due to point mutants, the observed significant binding affinity differences have previously been assessed with a focus on two α-helices located at the binding groove (α2) and the C-terminus (α3). Here, we employ a new computational approach to develop a generalized view on the molecular basis of PDZ3 binding selectivity and interaction communication for a set of point mutants of the protein (G330T, H372A, G330T-H372A) and its ligand (CRIPT named L1 and its T-2F variant L2) along with the wild type (WT). To analyze the dynamical aspects hidden in the conformations that are produced by molecular dynamics simulations, we utilize variations in community composition calculated based on the betweenness centrality measure from graph theory. We find that the highly charged N-terminus which is located far from the ligand has the propensity to share the same community with the ligand in the biologically functional complexes, indicating a distal segment might mediate the binding dynamics. N- and C-termini of PDZ3 share communities, and α3 acts as a hub for the whole protein by sustaining the communication with all structural segments, albeit being a trait not unique to the functional complexes. Moreover, α2 which lines the binding cavity frequently parts communities with the ligand and is not a controller of the binding but is rather a slave to the overall dynamics coordinated by the N-terminus. Thus, ligand binding fate in PDZ3 is traced to the population of community compositions extracted from dynamics despite the lack of significant conformational changes.

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 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.

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