Single-molecule fluorescence studies on the conformational change of the ABC transporter MsbA

Liu, Yanqing; Liu, Yue; He, Lingli; Zhao, Yongfang; Zhang, Xuejun C.

doi:10.1007/s41048-018-0057-z

Cited by 28 publications

(40 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…One key problem is achieving efficient fluorescence labeling of a functional transporter. And this may explain the rather limited number of published smFRET studies on ABC transporters labeled in the NBDs or TMDs [145], [146], [147], [148]. In the future, successful characterization of conformational dynamics may depend on alternative assays based on approaches that make quality sample preparation more readily achievable, such as the single label methods, protein induced fluorescence enhancement [149], [150] and quenching [151].…”

Section: Discussion and Outlookmentioning

confidence: 99%

An integrated transport mechanism of the maltose ABC importer

Mächtel

Narducci

Griffith

et al. 2019

Research in Microbiology

View full text Add to dashboard Cite

ATP-binding cassette (ABC) transporters use the energy of ATP hydrolysis to transport a large diversity of molecules actively across biological membranes. A combination of biochemical, biophysical, and structural studies has established the maltose transporter MalFGK2 as one of the best characterized proteins of the ABC family. MalF and MalG are the transmembrane domains, and two MalKs form a homodimer of nucleotide-binding domains. A periplasmic maltose-binding protein (MalE) delivers maltose and other maltodextrins to the transporter, and triggers its ATPase activity. Substrate import occurs in a unidirectional manner by ATP-driven conformational changes in MalK2 that allow alternating access of the substrate-binding site in MalF to each side of the membrane. In this review, we present an integrated molecular mechanism of the transport process considering all currently available information. Furthermore, we summarize remaining inconsistencies and outline possible future routes to decipher the full mechanistic details of transport by MalEFGK2 complex and that of related importer systems.

show abstract

Section: Discussion and Outlookmentioning

confidence: 99%

An integrated transport mechanism of the maltose ABC importer

Mächtel

Narducci

Griffith

et al. 2019

Research in Microbiology

View full text Add to dashboard Cite

show abstract

“…It is debated 9 , (i) if ATP hydrolysis is needed for reaching the outward facing and substrate-releasing state or (ii) if MgATP-induced NBD dimerisation alone leads to the outward-facing and substrate-releasing state. Experiments performed in whole cells 23 , along with crystal structures, EPR 71 and single molecule fluorescence studies 72 indicate that ATP binding shifts ABCB1 to its outward facing state. The affinity for substrates is lowest in the pre-hydrolytic ATP bound state, increases 2 fold in the post-hydrolytic vanadate trapped state, and 5 fold in the MgADP bound state after the release of P i and is highest in the absence of nucleotides 73,74 , indicating that the TMDs can assume more than two conformations.…”

Section: Discussionmentioning

confidence: 99%

Conversion of chemical to mechanical energy by the nucleotide binding domains of ABCB1

Szöllősi

Chiba

Stockner

2020

Sci Rep

View full text Add to dashboard Cite

P-glycoprotein (ABCB1) is an important component of barrier tissues that extrudes a wide range of chemically unrelated compounds. ABCB1 consists of two transmembrane domains forming the substrate binding and translocation domain, and of two cytoplasmic nucleotide binding domains (NBDs) that provide the energy by binding and hydrolyzing ATP. We analyzed the mechanistic and energetic properties of the NBD dimer via molecular dynamics simulations. We find that MgATP stabilizes the NBD dimer through strong attractive forces by serving as an interaction hub. The irreversible ATP hydrolysis step converts the chemical energy stored in the phosphate bonds of ATP into potential energy. Following ATP hydrolysis, interactions between the NBDs and the ATP hydrolysis products MgADP + Pi remain strong, mainly because Mg2+ forms stabilizing interactions with ADP and Pi. Despite these stabilizing interactions MgADP + Pi are unable to hold the dimer together, which becomes separated by avid interactions of MgADP + Pi with water. ATP binding to the open NBDs and ATP hydrolysis in the closed NBD dimer represent two steps of energy input, each leading to the formation of a high energy state. Relaxation from these high energy states occurs through conformational changes that push ABCB1 through the transport cycle.

show abstract

“…Finally, we should stress that measurements were performed in detergent micelles and it will be interesting to investigate whether the presence of a lipid bilayer (i.e. in a proteoliposome or a nanodisc), will affect the conformational dynamics (as in [32] for instance). The E histograms retrieved depend on the different conformations adopted by the protein but also on the interconversion rate.…”

Section: Discussionmentioning

confidence: 99%

Substrate binding modulates the conformational kinetics of the secondary multidrug transporter LmrP

Roth

Martens

Oene

et al. 2020

Preprint

View full text Add to dashboard Cite

The Major Facilitator Superfamily (MFS) is the largest family of secondary active membrane transporters and is found in all domains of Life. MFS proteins are known to adopt different conformational states, yet details on the interconversion rates are crucially needed to understand or target their transport mechanism. Here, we studied the proton/multidrug antiporter LmrP as a model system for antibiotic resistance development in bacteria. The conformational cycle of LmrP is triggered by the protonation of a network of specific amino acids, yet the role of the transported substrate in these transitions has been puzzling. To measure LmrP structure in real-time, we performed solution-based single-molecule Förster resonance energy transfer (smFRET) using a confocal microscope with direct alternating donor/acceptor excitation and multiparameter (intensity, lifetime, anisotropy) detection. Lowering pH from 8 to 5 triggered an overall conformational transition, corroborating that detergent solubilization allows studying the LmrP transport cycle using smFRET. Using a newly developed linear 3-state photon distribution analysis (PDA) model, we show that the apo protein interconverted between two structures at low rate (>>10 ms dwell time) at the cytosolic side while it interconverts dynamically BRIEF SUMMARYWe studied the conformational cycle of LmrP, a model for multidrug efflux pumps, using single-molecule Förster resonance energy transfer (smFRET). By following changes in FRET signal between different sets of positions, we specifically investigated how substrate binding modulates structural conversions between inward-open and outward-open states. Using a newly developed probabilistic analysis for describing sequential interconversion kinetics, we show that the apo protein slowly interconverts between defined states at the cytosolic and at the extracellular sides. Binding of the model substrate Hoechst33342 leads to an increase in conformational interconversions at the intracellular side while the extracellular side shows a drastic decrease in conversion, indicating a kinetic uncoupling between both sides. Remarkably, binding of roxithromycin, while also increasing interconversion on the intracellular side, did not slow the extracellular conversions. This indicates that multidrug pumps have evolved substrate-dependent transport mechanisms than enable transport of structurally diverse collection of substrates.

show abstract

Single-molecule fluorescence studies on the conformational change of the ABC transporter MsbA

Cited by 28 publications

References 40 publications

An integrated transport mechanism of the maltose ABC importer

An integrated transport mechanism of the maltose ABC importer

Conversion of chemical to mechanical energy by the nucleotide binding domains of ABCB1

Substrate binding modulates the conformational kinetics of the secondary multidrug transporter LmrP

Contact Info

Product

Resources

About