Structure and mechanotransmission mechanism of the MacB ABC transporter superfamily

MacB is an ABC transporter that collaborates with the MacA adaptor protein and TolC exit duct to drive efflux of antibiotics and enterotoxin STII out of the bacterial cell. Here we present the structure of ATP-bound MacB and reveal precise molecular details of its mechanism. The MacB transmembrane domain lacks a central cavity through which substrates could be passed, but instead conveys conformational changes from one side of the membrane to the other, a process we term mechanotransmission. Comparison of ATP-bound and nucleotide-free states reveals how reversible dimerization of the nucleotide binding domains drives opening and closing of the MacB periplasmic domains via concerted movements of the second transmembrane segment and major coupling helix. We propose that the assembled tripartite pump acts as a molecular bellows to propel substrates through the TolC exit duct, driven by MacB mechanotransmission. Homologs of MacB that do not form tripartite pumps, but share structural features underpinning mechanotransmission, include the LolCDE lipoprotein trafficking complex and FtsEX cell division signaling protein. The MacB architecture serves as the blueprint for understanding the structure and mechanism of an entire ABC transporter superfamily and the many diverse functions it supports.

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ATP-Induced Conformational Changes of Nucleotide-Binding Domains in an ABC Transporter. Importance of the Water-Mediated Entropic Force

The Journal of Physical Chemistry B ◽

10.1021/jp507930e ◽

2014 ◽

Vol 118 (44) ◽

pp. 12612-12620 ◽

Cited By ~ 15

Author(s):

Tomohiko Hayashi ◽

Shuntaro Chiba ◽

Yusuke Kaneta ◽

Tadaomi Furuta ◽

Minoru Sakurai

Keyword(s):

Abc Transporter ◽

Conformational Changes ◽

Nucleotide Binding ◽

Entropic Force ◽

Binding Domains

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Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

Open Biology ◽

10.1098/rsob.200406 ◽

2021 ◽

Vol 11 (4) ◽

Author(s):

Evelyn Ploetz ◽

Gea K. Schuurman-Wolters ◽

Niels Zijlstra ◽

Amarins W. Jager ◽

Douglas A. Griffith ◽

...

Keyword(s):

Ligand Binding ◽

Single Molecule ◽

Conformational Changes ◽

Protein Interactions ◽

Transmembrane Domain ◽

Substrate Binding ◽

Titration Calorimetry ◽

Uptake System ◽

Conformational States ◽

Binding Domains

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously.

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Conformational changes in the catalytically inactive nucleotide-binding site of CFTR

The Journal of General Physiology ◽

10.1085/jgp.201210954 ◽

2013 ◽

Vol 142 (1) ◽

pp. 61-73 ◽

Cited By ~ 19

Author(s):

László Csanády ◽

Csaba Mihályi ◽

Andras Szollosi ◽

Beáta Töröcsik ◽

Paola Vergani

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Nucleotide Binding ◽

Structural Rearrangement ◽

Nucleotide Binding Site ◽

Binding Domains ◽

Channel Opening ◽

Catalytically Active ◽

Opening And Closing ◽

Closing Rate

A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C→O1→O2→C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1. Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N6-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2×) transition O1→O2 and decreases the nonhydrolytic closing rate (transition O1→C) of CFTR mutants K1250A (∼4×) and E1371S (∼3×). Mutation H1348A also slowed (∼3×) the O1→O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A (∼3×) and E1371S (∼3×) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1→C and O1→O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.

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Insect ATP-Binding Cassette (ABC) Transporters: Roles in Xenobiotic Detoxification and Bt Insecticidal Activity

International Journal of Molecular Sciences ◽

10.3390/ijms20112829 ◽

2019 ◽

Vol 20 (11) ◽

pp. 2829 ◽

Cited By ~ 9

Author(s):

Chao Wu ◽

Swapan Chakrabarty ◽

Minghui Jin ◽

Kaiyu Liu ◽

Yutao Xiao

Keyword(s):

Abc Transporter ◽

Conformational Changes ◽

Abc Transporters ◽

Insecticidal Activity ◽

Atp Hydrolysis ◽

Atp Binding ◽

Bt Toxin ◽

Xenobiotic Detoxification ◽

Binding Domains ◽

Atp Binding Cassette

ATP-binding cassette (ABC) transporters, a large class of transmembrane proteins, are widely found in organisms and play an important role in the transport of xenobiotics. Insect ABC transporters are involved in insecticide detoxification and Bacillus thuringiensis (Bt) toxin perforation. The complete ABC transporter is composed of two hydrophobic transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). Conformational changes that are needed for their action are mediated by ATP hydrolysis. According to the similarity among their sequences and organization of conserved ATP-binding cassette domains, insect ABC transporters have been divided into eight subfamilies (ABCA–ABCH). This review describes the functions and mechanisms of ABC transporters in insecticide detoxification, plant toxic secondary metabolites transport and insecticidal activity of Bt toxin. With improved understanding of the role and mechanisms of ABC transporter in resistance to insecticides and Bt toxins, we can identify valuable target sites for developing new strategies to control pests and manage resistance and achieve green pest control.

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Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains

Journal of Bacteriology ◽

10.1128/jb.183.16.4761-4770.2001 ◽

2001 ◽

Vol 183 (16) ◽

pp. 4761-4770 ◽

Cited By ~ 18

Author(s):

Juan M. Falcón-Pérez ◽

Mónica Martı́nez-Burgos ◽

Jesús Molano ◽

Marı́a J. Mazón ◽

Pilar Eraso

Keyword(s):

Atpase Activity ◽

Abc Transporter ◽

Transmembrane Domain ◽

Substrate Binding ◽

Nucleotide Binding ◽

Intramolecular Interactions ◽

Phenotypic Characterization ◽

Atp Binding ◽

Binding Domains ◽

Atp Binding Cassette

ABSTRACT The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V max of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.

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Conformational changes opening and closing the CFTR chloride channel: Insights from cysteine scanning mutagenesis

Biochemistry and Cell Biology ◽

10.1139/bcb-2014-0038 ◽

2014 ◽

Vol 92 (6) ◽

pp. 481-488 ◽

Cited By ~ 11

Author(s):

Yassine El Hiani ◽

Paul Linsdell

Keyword(s):

Cystic Fibrosis ◽

Conformational Changes ◽

Large Family ◽

Chloride Ion ◽

Channel Structure ◽

Patch Clamp Recording ◽

Binding Domains ◽

Substituted Cysteine Accessibility ◽

Cysteine Scanning Mutagenesis ◽

Opening And Closing

Cystic fibrosis, the most common lethal genetic disease affecting young people in North America, is caused by failure of the chloride ion channel known as CFTR (cystic fibrosis transmembrane conductance regulator). CFTR belongs to the large family of ATP-binding cassette (ABC) membrane transporters. In CFTR, ATP-driven events at the nucleotide-binding domains (NBDs) open and close a gate that controls chloride permeation. However, the conformational changes concomitant with opening and closing of the CFTR gate are unknown. Diverse techniques including substituted cysteine accessibility method, disulfide cross-linking, and patch-clamp recording have been used to explore CFTR channel structure. Here, we consider the architecture of both the open and the closed CFTR channel. We review how CFTR channel structure changes between the closed and the open channel conformations and portray the relative function of both cytoplasmic and vestigial gates during the gating cycle. Understanding how the CFTR channel gates chloride permeation is central for understanding how CFTR defects lead to CF. Such knowledge opens the door for novel ways to maximize CFTR channel activity in a CF setting.

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ATP and Substrate Binding Regulates Conformational Changes of Human Peroxisomal ABC Transporter ALDP

10.21203/rs.3.rs-1002426/v1 ◽

2021 ◽

Author(s):

Lin Tang ◽

Chao Xiong ◽

Lina Jia ◽

Ming-He Shen ◽

Wei-Xi Xiong ◽

...

Keyword(s):

Electron Microscopy ◽

Fatty Acids ◽

Conformational Changes ◽

Transmembrane Domain ◽

Long Chain Fatty Acids ◽

Variable Size ◽

Binding Domains ◽

Cryo Electron Microscopy ◽

Peroxisome Membrane ◽

Long Chain

Abstract The malfunction of ABCD1 causes X-linked adrenoleukodystrophy (X-ALD), a rare neurodegenerative disease that affect all tissues in human. Residing in the peroxisome membrane, ABCD1 plays a role in the translocation of very long chain fatty acids (VLCFA) for their damage by β-oxidation. Here, we present five Cryo-Electron microscopy structures of ABCD1 in four conformational states. Combined with functional analysis, we found that substrate and ATP trigger the closing of two nucleotide binding domains (NBDs) over a distance of 40 Å and the rearrangement of the transmembrane domains. Each of the three inward-facing structure of ABCD1 has a vestibule opens to cytosol with variable size. Furthermore, the structure of ABCD1 in the outward-facing state supports that ATP molecules pull the two NBDs together and open the transmembrane domain to the peroxisomal lumen for substrate release. The five structures provide a snapshot of substrate transporting cycle and mechanistic implications for disease-causing mutations.

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Probing native metal ion association sites through quenching of fluorophores in the nucleotide-binding domains of the ABC transporter MsbA

Biochemical Journal ◽

10.1042/bcj20161051 ◽

2017 ◽

Vol 474 (12) ◽

pp. 1993-2007 ◽

Cited By ~ 1

Author(s):

Daiki Tatsumi ◽

Kei Nanatani ◽

Yuto Koike ◽

Kiyoto Kamagata ◽

Satoshi Takahashi ◽

...

Keyword(s):

Abc Transporter ◽

Conformational Changes ◽

Metal Ion ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Ion Association ◽

Nucleotide Binding ◽

Small Scale ◽

Collisional Quenching ◽

Binding Domains

ATP-binding cassette (ABC) transporters are ubiquitously present in prokaryotic and eukaryotic cells. Binding of ATP to the nucleotide-binding domains (NBDs) elicits major conformational changes of the transporters resulting in the transport of the substrate across the membrane. The availability of a crystal structure of the NBDs enabled us to elucidate the local structure and small-scale dynamics in the NBDs. Here, we labeled the ABC transporter MsbA, a homodimeric flippase from Escherichia coli, with a fluorescent probe, Alexa532, within the NBDs. ATP application elicited collisional quenching, whereas no quenching was observed after the addition of ATP analogs or ATP hydrolysis inhibitors. The Alexa532-conjugated MsbA variants exhibited transition metal ion Förster resonance energy transfer (tmFRET) after the addition of Ni2+, and ATP decreased this Ni2+-mediated FRET of the NBDs. Structure modeling developed from crystallographic data and examination of tmFRET measurements of MsbA variants in the absence of ATP revealed the presence of metal ion-associated pockets (MiAPs) in the NBDs. Three histidines were predicted to participate in chelating Ni2+ in the two possible MiAPs. Performing histidine-substitution experiments with the NBDs showed that the dissociation constant for Ni2+ of MiAP2 was smaller than that of MiAP1. The structural allocation of the MiAPs was further supported by showing that the addition of Cu2+ resulted in higher quenching than Ni2+. Taken together, the present study showed that the NBDs contain two native binding sites for metal ions and ATP addition affects the Ni2+-binding activity of the MiAPs.

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The dynamic action of SecA during the initiation of protein translocation

Biochemical Journal ◽

10.1042/bj20121314 ◽

2013 ◽

Vol 449 (3) ◽

pp. 695-705 ◽

Cited By ~ 28

Author(s):

Vicki A. M. Gold ◽

Sarah Whitehouse ◽

Alice Robson ◽

Ian Collinson

Keyword(s):

Conformational Changes ◽

Large Scale ◽

Protein Transport ◽

Protein Translocation ◽

Cross Linking ◽

High Affinity ◽

Binding Domains ◽

Alternative Position ◽

Opening And Closing

The motor ATPase SecA drives protein secretion through the bacterial Sec complex. The PPXD (pre-protein cross-linking domain) of the enzyme has been observed in different positions, effectively opening and closing a clamp for the polypeptide substrate. We set out to explore the implicated dynamic role of the PPXD in protein translocation by examining the effects of its immobilization, either in the position occupied in SecA alone with the clamp held open or when in complex with SecYEG with the clamp closed. We show that the conformational change from the former to the latter is necessary for high-affinity association with SecYEG and a corresponding activation of ATPase activity, presumably due to the PPXD contacting the NBDs (nucleotide-binding domains). In either state, the immobilization prevents pre-protein transport. However, when the PPXD was attached to an alternative position in the associated SecYEG complex, with the clamp closed, the transport capability was preserved. Therefore large-scale conformational changes of this domain are required for the initiation process, but not for translocation itself. The results allow us to refine a model for protein translocation, in which the mobility of the PPXD facilitates the transfer of pre-protein from SecA to SecYEG.

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Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

10.1101/2020.12.19.423572 ◽

2020 ◽

Author(s):

Evelyn Ploetz ◽

Gea K. Schuurman-Wolters ◽

Niels Zijlstra ◽

Amarins W. Jager ◽

Douglas A. Griffith ◽

...

Keyword(s):

Crystal Structure ◽

Ligand Binding ◽

Conformational Changes ◽

Transmembrane Domain ◽

Substrate Binding ◽

List Type ◽

Binding Affinities ◽

Proof Of Concept ◽

Conformational States ◽

Binding Domains

ABSTRACTThe ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid, and asparagine in Gram-positive bacteria. It features two extracytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from L. lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster-resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE-FRET to monitor protein-protein interactions and conformational states simultaneously.HIGHLIGHTSResolved crystal structure of tandem SBD1-2 of GlnPQ from Lactococcus lactisConformational states and ligand binding affinities of individual domains SBD1 and SBD2 are similar to tandem SBD1-2No cooperative effects are seen for different ligands for SBDs in the tandemProof of concept experiments show that PIFE-FRET can monitor SBD conformations and protein-protein interaction simultaneously

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