Structural Basis of Energy Transduction in the Transport Cycle of MsbA

Dong, Jinhui; Yang, Gaoqiang; Mchaourab, Hassane S.

doi:10.1126/science.1106592

Cited by 152 publications

(160 citation statements)

References 31 publications

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“…These data were questioned as a detergent artifact mistaken for dynamic NBDs. DEER data of ABC transporters in liposomes and bicelles show broad lines for apo NBDs (Dong et al , 2005; Borbat et al , 2007; Zou et al , 2009; Bountra et al , 2017), further suggesting that in the absence of nucleotides the NBDs can have different degrees of disengagement. Upon nucleotide binding, the NBDs dimerize and display nearly identical distances across different transporters (Ward et al , 2007; Lin et al , 2015; Perez et al , 2015; Bountra et al , 2017).…”

Section: Discussion and Outlookmentioning

confidence: 99%

“…All ABC transporters share a common architecture consisting of a transmembrane domain (TMD) for substrate recognition and transport, and a nucleotide‐binding domain (NBD) that converts the chemical energy of ATP into conformational changes for transport (Beis, 2015). The structures of several homodimeric (Dawson & Locher, 2006; Ward et al , 2007; Perez et al , 2015) and heterodimeric ABC transporters (Hohl et al , 2012; Noll et al , 2017) revealed distinct conformations and suggest, in combination with biophysical studies (e.g., EPR and NMR; Dong et al , 2005; Zou et al , 2009; Bountra et al , 2017; Timachi et al , 2017; Barth et al , 2018), that they undergo large conformational changes during transport. Their complex architecture is, however, a fundamental hurdle to fully understand the coupling between conformational changes, substrate binding, ATP binding and hydrolysis, and transport.…”

Section: Introductionmentioning

confidence: 99%

“…ABC transporters that utilize the alternating access mechanism undergo large conformational changes during the transport cycle suggested by crystal structures (Ward et al , 2007) and EPR‐based Double Electron Electron Resonance (DEER) measurements (Dong et al , 2005; Borbat et al , 2007; Mishra et al , 2014) of the lipid A flippase MsbA from Escherichia coli . Although the DEER measurements proposed that ATP triggers the opening of the TMD to the periplasmic side for release of the substrate, it is unclear how precisely these changes are coupled to ATP binding and hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

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Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

et al. 2018

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ABC transporters utilize ATP for export processes to provide cellular resistance against toxins, antibiotics, and harmful metabolites in eukaryotes and prokaryotes. Based on static structure snapshots, it is believed that they use an alternating access mechanism, which couples conformational changes to ATP binding (outward‐open conformation) and hydrolysis (inward‐open) for unidirectional transport driven by ATP. Here, we analyzed the conformational states and dynamics of the antibacterial peptide exporter McjD from Escherichia coli using single‐molecule Förster resonance energy transfer (smFRET). For the first time, we established smFRET for an ABC exporter in a native‐like lipid environment and directly monitor conformational dynamics in both the transmembrane‐ (TMD) and nucleotide‐binding domains (NBD). With this, we unravel the ligand dependences that drive conformational changes in both domains. Furthermore, we observe intrinsic conformational dynamics in the absence of ATP and ligand in the NBDs. ATP binding and hydrolysis on the other hand can be observed via NBD conformational dynamics. We believe that the progress made here in combination with future studies will facilitate full understanding of ABC transport cycles.

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Section: Discussion and Outlookmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

et al. 2018

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“…[16][17][18][19] High-resolution structures and NMR, EPR, and electron microscopy data strongly suggest that ABC exporters undergo large movements during substrate efflux, in particular upon ATP binding and/or hydrolysis. [20][21][22][23][24][25][26][27] Although structure determinations and the large body of experimental data obtained on ABC transporters have shed light on the transport mechanism, the answers to some questions remain elusive: what are the conformational changes occurring upon substrate binding and ATP binding and/or hydrolysis, and how do these changes relate to solute translocation? Molecular dynamics (MD) simulations are a means of exploring the conformational dynamics of proteins with an extreme level of detail as regards atomic motions.…”

Section: Introductionmentioning

confidence: 99%

Dynamics and Structural Changes Induced by ATP Binding in SAV1866, a Bacterial ABC Exporter

et al. 2010

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Multidrug transporters of the ATP-binding cassette family export a wide variety of compounds across membranes in both prokaryotes and eukaryotes, using ATP hydrolysis as energy source. Several of these membrane proteins are of clinical importance. Although biochemical and structural studies have provided insights into the mechanism underlying substrate transport, many key questions subsist regarding the molecular and structural nature of this mechanism. In particular, the detailed conformational changes occurring during the catalytic cycle are still elusive. We explored the conformational changes occurring upon ATP/Mg 2+ binding using molecular dynamics simulations starting from the nucleotide-bound structure of SAV1866 embedded in an explicit lipid bilayer. The removal of nucleotide revealed a major rearrangement in the outer membrane leaflet portion of the transmembrane domain (TMD) resulting in the closure of the central cavity at the extracellular side. This closure is similar to that observed in the crystal nucleotide-free structures. The interface of the nucleotide-binding domain dimer (NDB) is significantly more hydrated in the nucleotide-free trajectory though it is not disrupted. This finding suggests that the TMD closure could occur as a first step preceding the dissociation of the dimer. The transmission pathway of the signal triggered by the removal of ATP/Mg 2+ mainly involves the conserved Q-loop and X-loop as well as TM6.

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“…For this purpose, spin label probes were introduced one at a time along the EmrE sequence (residues 3-110). EPR spectroscopy was used to characterize spin label dynamics, accessibility to the lipid and water phases, as well as pairwise short range proximity across the dimer interface (22)(23)(24)(25). Changes in the EPR constraints upon substrate binding reveal movements in TM1 and -2 and ordering of TM3 and the adjacent loop linking it to TM4.…”

mentioning

confidence: 99%

Structure, Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

Amadi¹,

Koteiche²,

Mishra

et al. 2010

Journal of Biological Chemistry

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EmrE, a member of the small multidrug transporters superfamily, extrudes positively charged hydrophobic compounds out of Escherichia coli cytoplasm in exchange for inward movement of protons down their electrochemical gradient. Although its transport mechanism has been thoroughly characterized, the structural basis of energy coupling and the conformational cycle mediating transport have yet to be elucidated. In this study, EmrE structure in liposomes and the substrate-induced conformational changes were investigated by systematic spin labeling and EPR analysis. Spin label mobilities and accessibilities describe a highly dynamic ligand-free (apo) conformation. Dipolar coupling between spin labels across the dimer reveals at least two spin label populations arising from different packing interfaces of the EmrE dimer. One population is consistent with antiparallel arrangement of the monomers, although the EPR parameters suggest deviations from the crystal structure of substrate-bound EmrE. Resolving these discrepancies requires an unusual disposition of TM3 relative to the membrane-water interface and a kink in its backbone that enables bending of its C-terminal part. Binding of the substrate tetraphenylphosphonium changes the environment of spin labels and their proximity in three transmembrane helices. The underlying conformational transition involves repacking of TM1, tilting of TM2, and changes in the backbone configurations of TM3 and the adjacent loop connecting it to TM4. A dynamic apo conformation is necessary for the polyspecificity of EmrE allowing the binding of structurally diverse substrates. The flexibility of TM3 may play a critical role in movement of substrates across the membrane.One mechanism of multidrug resistance involves the active extrusion of toxic molecules out of the cell by dedicated membrane transporters. In prokaryotes, five superfamilies of transporters handle vectorial drug traffic moving energetically uphill against substrate concentration gradients (1). The thermodynamics of the problem are rendered favorable through coupling of substrate movement to the direct use of ATP energy or the discharge of electrochemical ion gradients. Small multidrug resistance transporters are the smallest bacterial transporters with four predicted transmembrane ␣-helices and no significant extramembrane domain (2, 3). They function as dimers coupling translocation of positively charged hydrophobic substrates out of the cell to the inward movement of protons.Much of the current mechanistic understanding of small multidrug resistance transporters has emerged from seminal studies in the laboratory of Schuldiner and co-workers (4 -7)defining fundamental steps in the transport cycle of Escherichia coli EmrE. Protons and substrates share a common binding site organized around the absolutely conserved, membrane-embedded, glutamate 14 in transmembrane (TM) 3 helix 1 (8). Binding of positively charged hydrophobic substrates is coordinated by the Glu-14 side chain and stabilized by interactions with aromatic res...

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Structural Basis of Energy Transduction in the Transport Cycle of MsbA

Cited by 152 publications

References 31 publications

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

Dynamics and Structural Changes Induced by ATP Binding in SAV1866, a Bacterial ABC Exporter

Structure, Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

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