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Sequential Action of MalE and Maltose Allows Coupling ATP Hydrolysis to Translocation in the MalFGK2 Transporter*

  • Huan Bao
    Footnotes
    Affiliations
    Departments of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada
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  • Kush Dalal
    Footnotes
    Affiliations
    Departments of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada
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  • Eric Cytrynbaum
    Affiliations
    Departments of Mathematics, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada
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  • Franck Duong
    Correspondence
    To whom correspondence should be addressed: Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T1Z3, Canada. Tel.: 604-822-5975; Fax: 604-822-522
    Affiliations
    Departments of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada
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  • Author Footnotes
    * This work was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR) (to F. D. and E. C.) and a Four-Year Fellowship from the University of British Columbia (to H. B.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Both authors contributed equally to this work.
    2 Present address: Vancouver Prostate Centre, University of British Columbia, Vancouver, British Columbia V6H 3Z6, Canada.
Open AccessPublished:September 03, 2015DOI:https://doi.org/10.1074/jbc.M115.671826
      ATP-binding cassette (ABC) transporters have evolved an ATP-dependent alternating-access mechanism to transport substrates across membranes. Despite important progress, especially in their structural analysis, it is still unknown how the substrate stimulates ATP hydrolysis, the hallmark of ABC transporters. In this study, we measure the ATP turnover cycle of MalFGK2 in steady and pre-steady state conditions. We show that (i) the basal ATPase activity of MalFGK2 is very low because the cleavage of ATP is rate-limiting, (ii) the binding of open-state MalE to the transporter induces ATP cleavage but leaves release of Pi limiting, and (iii) the additional presence of maltose stimulates release of Pi, and therefore increases the overall ATP turnover cycle. We conclude that open-state MalE stabilizes MalFGK2 in the outward-facing conformation until maltose triggers return to the inward-facing state for substrate and Pi release. This concerted action explains why ATPase activity of MalFGK2 depends on maltose, and why MalE is essential for transport.

      Introduction

      ABC
      The abbreviations used are: ABC
      ATP-binding cassette
      TMD
      transmembrane domain
      NBD
      nucleotide-binding domain
      TNP-ATP
      2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate
      MESG
      2-amino-6-mercapto-7-methylpurine riboside.
      transporters are widespread and universally conserved in nature. They consist of at least two transmembrane domains (TMDs) that form the substrate passageway, as well as two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP (
      • Higgins C.F.
      ABC transporters: from microorganisms to man.
      ). In bacteria, ABC transporters are essential for pumping in nutrients and for pumping out toxic compounds. In humans, they perform equally important functions; their deregulation is often linked to diseases (
      • Cole S.P.
      Multidrug resistance protein 1 (MRP1, ABCC1), a “multitasking” ATP-binding cassette (ABC) transporter.
      ,
      • Cutting G.R.
      Cystic fibrosis genetics: from molecular understanding to clinical application.
      ,
      • Seyffer F.
      • Tampé R.
      ABC transporters in adaptive immunity.
      ). Understanding the working mechanism of ABC transporters is therefore central to biomedical research. Intensive crystallographic work has led to a general model of action: ATP drives the association of the NBDs and the transition of the TMDs from an inward-facing to an outward-facing conformation. Hydrolysis of ATP leads to the dissociation of the NBDs, and thus returns the TMDs to their initial inward-facing conformation. This alternating exposure of the TMDs on either side of the membrane, termed the alternating-access mechanism, allows substrate binding, transport, and release, while preserving membrane impermeability (
      • Procko E.
      • O'Mara M.L.
      • Bennett W.F.D.
      • Tieleman D.P.
      • Gaudet R.
      The mechanism of ABC transporters: general lessons from structural and functional studies of an antigenic peptide transporter.
      ,
      • Locher K.P.
      Review. Structure and mechanism of ATP-binding cassette transporters.
      ,
      • Chen J.
      Molecular mechanism of the Escherichia coli maltose transporter.
      ,
      • ter Beek J.
      • Guskov A.
      • Slotboom D.J.
      Structural diversity of ABC transporters.
      ).
      An important unresolved question for ABC transporters is the mechanism by which substrate stimulates ATP hydrolysis. The degree of stimulation varies, but the characteristic is common to the family (
      • Davidson A.L.
      • Shuman H.A.
      • Nikaido H.
      Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins.
      ,
      • Ames G.F.
      • Liu C.E.
      • Joshi A.K.
      • Nikaido K.
      Liganded and unliganded receptors interact with equal affinity with the membrane complex of periplasmic permeases, a subfamily of traffic ATPases.
      ,
      • Landmesser H.
      • Stein A.
      • Blüschke B.
      • Brinkmann M.
      • Hunke S.
      • Schneider E.
      Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium.
      ,
      • Patzlaff J.S.
      • van der Heide T.
      • Poolman B.
      The ATP/substrate stoichiometry of the ATP-binding cassette (ABC) transporter OpuA.
      ,
      • Borths E.L.
      • Poolman B.
      • Hvorup R.N.
      • Locher K.P.
      • Rees D.C.
      In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake.
      ,
      • Eckford P.D.
      • Sharom F.J.
      Functional characterization of Escherichia coli MsbA: interaction with nucleotides and substrates.
      ). The crystal structures of ABC exporters suggest that binding of the substrate to the inward-facing TMDs provokes pre-closure of the NBDs: a conformational state termed the primed or pre-hydrolytic state (
      • Doshi R.
      • van Veen H.W.
      Substrate binding stabilizes a pre-translocation intermediate in the ATP-binding cassette transport protein MsbA.
      ,
      • Ward A.
      • Reyes C.L.
      • Yu J.
      • Roth C.B.
      • Chang G.
      Flexibility in the ABC transporter MsbA: alternating access with a twist.
      ,
      • Jones P.M.
      • O'Mara M.L.
      • George A.M.
      ABC transporters: a riddle wrapped in a mystery inside an enigma.
      ). This pre-closure of the NBDs in turn facilitates the movement of the TMDs to the outward-facing conformation, and eventually the release of the substrate on the trans-side of the membrane. Upon hydrolysis of ATP, the NBDs dissociate and the TMDs return to their initial inward-facing state. As a consequence, the binding of the substrate to the transporter augments the hydrolysis of ATP. This proposed mechanism of action is supported by structural and functional analysis on ABC exporters (
      • Doshi R.
      • van Veen H.W.
      Substrate binding stabilizes a pre-translocation intermediate in the ATP-binding cassette transport protein MsbA.
      ,
      • Choudhury H.G.
      • Tong Z.
      • Mathavan I.
      • Li Y.
      • Iwata S.
      • Zirah S.
      • Rebuffat S.
      • van Veen H.W.
      • Beis K.
      Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state.
      ,
      • Lee J.Y.
      • Yang J.G.
      • Zhitnitsky D.
      • Lewinson O.
      • Rees D.C.
      Structural basis for heavy metal detoxification by an Atm1-type ABC exporter.
      ). In the case of ABC importers, however, the stimulatory action of the substrate needs to occur via a different mechanism for two reasons: (i) the TMDs must reach their outward-facing conformation in order for the substrate to enter the transport pathway and (ii) the transport and ATPase activities depend on a peripheral substrate-binding protein. Because the binding protein itself stimulates the ATPase cycle, the role of the substrate is furthermore obscure (
      • Davidson A.L.
      • Shuman H.A.
      • Nikaido H.
      Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins.
      ,
      • Ames G.F.
      • Liu C.E.
      • Joshi A.K.
      • Nikaido K.
      Liganded and unliganded receptors interact with equal affinity with the membrane complex of periplasmic permeases, a subfamily of traffic ATPases.
      ,
      • Borths E.L.
      • Poolman B.
      • Hvorup R.N.
      • Locher K.P.
      • Rees D.C.
      In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake.
      ). The binding protein exists in equilibrium between open and closed states, but the binding of the substrate favors the closed state. It is therefore often proposed that binding of the closed-liganded protein induces the closure of the NBDs and therefore cleavage of ATP (e.g. Ref.
      • Oldham M.L.
      • Chen J.
      Crystal structure of the maltose transporter in a pretranslocation intermediate state.
      ).
      Here, we analyze the MalFGK2 transporter to better understand the ATP hydrolytic cycle, as well as its dependence on MalE and maltose. This kinetic analysis is made possible by incorporating MalFGK2 into nanodiscs. Previous analyses show that MalFGK2 in nanodiscs behaves as it does in proteoliposomes: (i) the basal ATPase activity of MalFGK2 is naturally low; (ii) unliganded MalE stimulates the ATPase activity by ∼4-fold (to ∼40 nmol/min/mg); and (iii) the presence of MalE and maltose stimulates ATP hydrolysis by ∼40-fold (∼400 nmol/min/min) (
      • Davidson A.L.
      • Shuman H.A.
      • Nikaido H.
      Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins.
      ,
      • Bao H.
      • Dalal K.
      • Wang V.
      • Rouiller I.
      • Duong F.
      The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.
      ). Using a transient kinetic method, we show that open-state MalE binds to the outward-facing transporter to trigger the cleavage of ATP. Maltose subsequently facilitates the release of hydrolyzed Pi. These findings form the basis for understanding the transport reaction.

      Experimental Procedures

      Materials

      MalFGK2, MalE, and EIIAGlc proteins were overexpressed and purified as before (
      • Bao H.
      • Duong F.
      Phosphatidylglycerol directs binding and inhibitory action of EIIAGlc protein on the maltose transporter.
      ,
      • Bao H.
      • Duong F.
      Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation.
      ). Reconstitution of MalFGK2 into proteoliposomes was carried out as described (
      • Bao H.
      • Dalal K.
      • Wang V.
      • Rouiller I.
      • Duong F.
      The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.
      ). Nanodiscs were prepared at a molar ratio MalFGK2:MSP:lipids (where MSP is membrane scaffold protein) of 1:3:600 (
      • Bao H.
      • Dalal K.
      • Wang V.
      • Rouiller I.
      • Duong F.
      The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.
      ). Lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG) were purchased from Avanti Polar lipids. Radiolabeled nucleotides [α-32P]ATP (25 Ci/mmol) and [γ-32P]ATP (800 Ci/mmol) were obtained from MP Biomedicals. The EnzChek kit was from Molecular Probes (Invitrogen). Kinase-Glo reagent was from Promega. All other chemicals, including, pyruvate kinase (catalogue number P9136), lactate dehydrogenase (catalogue number L38888), TNP-ATP, and polyethylenimine cellulose TLC plates, were supplied by Sigma.

      ATPase Assays

      Steady state ATPase assays were performed at the indicated temperature using 1 μm MalFGK2 nanodiscs, 10 μm MalE, 1 mm maltose, and 1 mm [α-32P]ATP in ATPase buffer (50 mm Tris-HCl, pH 7.5; 5 mm MgCl2). To determine the Km value of MalFGK2 for ATP, experiments were carried out at 25 °C with increasing concentrations of [α-32P]ATP. For single turnover ATPase assays, reactions were carried out at the indicated temperature using 20 μm MalFGK2 nanodiscs, 100 μm MalE, 1 mm maltose, and 1 μm [α-32P]ATP in ATPase buffer. The quantity of ATP hydrolyzed was determined by TLC as described previously (
      • Bao H.
      • Duong F.
      Phosphatidylglycerol directs binding and inhibitory action of EIIAGlc protein on the maltose transporter.
      ), followed by autoradiography and density scanning on a Typhoon imager.

      Nucleotide Binding Assay to MalFGK2

      Binding of TNP-ATP to MalFGK2 was performed as described previously (
      • Bao H.
      • Duong F.
      Phosphatidylglycerol directs binding and inhibitory action of EIIAGlc protein on the maltose transporter.
      ). The fluorescence data were plotted as a function of TNP-ATP concentration and fit to Equation 1,
      F=Fmax×[L][L]+Kd
      (Eq. 1)


      where Fmax is the maximal subtracted fluorescence at saturating amount of TNP-ATP and Kd is the equilibrium dissociation constant of TNP-ATP for MalFGK2. To determine the affinity of MalFGK2 for ATP, TNP-ATP (80 μm) was incubated with MalFGK2 nanodiscs (2 μm) for 5 min at 25 °C and then titrated with an increased amount of ATP. The fluorescence data were fit to Equation 2,
      F=F0×Ki,app[I]+Ki,app+[I][I]+Ki,app
      (Eq. 2)


      in which F0 is the fluorescence value in the absence of ATP, F1 is the fluorescence value in the presence of saturating amount of ATP, [I] is the ATP concentration, and Ki,app is the apparent inhibition constant of ATP at the specified amount of TNP-ATP. The Kd,app of ATP for MalFGK2 is calculated from Ki,app by Equation 3,
      Ki,app=Kd,app×(1+[L]Kd)
      (Eq. 3)


      in which [L] is the TNP-ATP concentration, and Kd is the dissociation constant of TNP-ATP for MalFGK2.

      Stopped-flow Experiments

      Measurements were performed at 25 °C using an SX-18MV stopped-flow apparatus (Applied Photophysics Ltd., Leatherhead, UK). Data collection was carried out with 5 μm MalFGK2 nanodiscs, 50 μm MalE, 1 mm maltose, and 1 mm ATP in ATPase buffer. Release of ADP was measured using the pyruvate kinase/lactate dehydrogenase enzyme-linked assay (
      • Robson A.
      • Gold V.A.M.
      • Hodson S.
      • Clarke A.R.
      • Collinson I.
      Energy transduction in protein transport and the ATP hydrolytic cycle of SecA.
      ). Absorbance data were collected at 340 nm, and quantity of ADP produced was calculated using the extinction coefficient of NADH (6,220 m−1 cm−1). Release of phosphate was measured using the EnzChek kit. Absorbance data were collected at 360 nm, and the quantity of phosphate produced was calculated using the extinction coefficient of MESG (11,000 m−1 cm−1). Kinetic data of phosphate release were fit to a simple linear regression model, whereas kinetic data of ADP release were fit to Equation 4,
      A=A0(1ekburst)+klinear
      (Eq. 4)


      where A is the amount of calculated ADP, A0 is the amount of ADP in the exponential phase, kburst is the rate constant for the burst phase, and klinear is the rate constant for the slower linear phase.

      Quenched-flow Experiments

      The kinetics of ATP cleavage were measured at 25 °C using a QFM-400 quenched-flow apparatus (Bio-Logic SAS). Experiments were carried out using 25 μm MalFGK2 nanodiscs, 100 μm MalE, 1 mm maltose, and 1 mm ATP in ATPase buffer. At the indicated time points, reactions were quenched by buffer A (50 mm Tris-HCl, pH 7.5; 50 mm EDTA). The amount of ATP was quantified with Kinase-Glo reagent, and the data were fit to Equation 5,
      A=(A0B)ekburstklineart+B
      (Eq. 5)


      where A0 is the amount of ATP before the initiation of the reaction, B is the amount of ATP at the end of the burst phase of the reaction, kburst is the rate constant for the burst phase, and klinear is the rate constant for the slower linear phase.

      Other Methods

      For TLC and centrifugal gel filtration assays, MalFGK2 nanodiscs (20 μm), MalE (75 μm), and maltose (1 mm) were incubated with [α-32p]ATP or [γ-32p]ATP (100 μm) at 4 °C for 10 min. Samples were subjected to centrifugal gel filtration and analyzed by TLC.

      Results

      Binding of ATP to MalK Is Independent of MalE and Maltose

      To understand how MalE and maltose accelerate the ATP hydrolytic cycle of MalFGK2, we performed an Arrhenius analysis of its ATPase activity. The data reveal that MalE alone lowers the ATPase activation energy state of the transporter by ∼20%; however, maltose alone has little effect (Fig. 1a). This analysis led us conclude that the stimulatory action of MalE occurs before that of maltose.
      Figure thumbnail gr1
      FIGURE 1Binding of ATP to MalFGK2 is not stimulated by MalE and maltose. a, left panel, the Kcat values (pmol of Pi/pmol of MalFGK2/min) are determined as a function of temperature. The y axis represents the natural logarithm of the Kcat values [lnKcat], and the x axis represents the inversed temperature values (1/T) expressed in Kelvin. Right panel, the activation energy Ea was calculated from the slope of the linear plot, where the slope = −Ea/R and R = 8.31451 J K−1 mol−1. b, equilibrium titration of TNP-ATP binding to MalFGK2. The MalFGK2 complex was incubated with TNP-ATP, in the presence or absence of MalE and maltose. Fluorescence data were plotted against concentration of TNP-ATP. Data were fit to and summarized in (mean ± S.D., n = 3 independent replicates). c, MalFGK2 ATPase activity as a function of ATP concentrations, in the presence or absence of MalE and maltose. Data were fit to the Michaelis-Menten equation, and values are reported in (mean ± S.D., n = 3 independent replicates).
      To determine whether MalE affects the binding of ATP to the transporter, we employed an ATP analog labeled with trinitrophenyl (TNP) (
      • Poolman B.
      • Doeven M.K.
      • Geertsma E.R.
      • Biemans-Oldehinkel E.
      • Konings W.N.
      • Rees D.C.
      Functional analysis of detergent-solubilized and membrane-reconstituted ATP-binding cassette transporters.
      ). The quantum yield of TNP-ATP increases upon binding to MalK, yet the increase is independent of both MalE and maltose (Fig. 1b). The apparent Kd for ATP, measured by competitive replacement of TNP-ATP, is ∼210 μm (Table 1). This value is similar to the Km derived from the ATPase measurements (∼230 μm; Fig. 1c). When the measurements were repeated with MalE and maltose, no significant change in affinity was detected (Table 1), indicating that MalE and maltose exert their stimulatory action after ATP is bound to the transporter.
      TABLE 1Affinities of MalFGK2 for nucleotides
      Km for ATPKd for TNP-ATPKd,app for ATP
      μmμmμm
      MalFGK2233 ± 429 ± 1211 ± 23
      MalFGK2, MalE274 ± 3210 ± 2194 ± 22
      MalFGK2, MalE, maltose283 ± 419 ± 1224 ± 33

      MalE Stimulates the ATP Cleavage Reaction

      We next determined the capacity of MalE and maltose to accelerate the cleavage of ATP under two different conditions: (i) when MalFGK2 is present in 20-fold molar excess over the nucleotide, so that only a single round of ATP hydrolysis is possible (Fig. 2a), and (ii) under steady state conditions, where the nucleotide is present in 1000-fold excess over MalFGK2, so that multiple rounds of ATP hydrolysis can occur (Fig. 2b). MalFGK2 was incubated with [α-32P]ATP, and ATP hydrolysis was detected by thin layer chromatography. Under single turnover conditions, ∼50% of total ATP is hydrolyzed within 1 min, at 4 °C or at 37 °C. Strikingly, under the same conditions, the addition of MalE results in ∼100% cleavage of ATP in a similar time period. In contrast, much less cleavage was detected when MalE and maltose are present together when compared with MalE alone (Fig. 2a). The latter result is not surprising because closed-liganded MalE has very low affinity for the transporter (
      • Telmer P.G.
      • Shilton B.H.
      Insights into the conformational equilibria of maltose-binding protein by analysis of high affinity mutants.
      ,
      • Gould A.D.
      • Telmer P.G.
      • Shilton B.H.
      Stimulation of the maltose transporter ATPase by unliganded maltose binding protein.
      ,
      • Bao H.
      • Duong F.
      Discovery of an auto-regulation mechanism for the maltose transporter MalFGK2.
      ). Accordingly, the inhibitory effect of maltose is enhanced with the mutant MalE-DW, and this inhibitory effect is not observed with the mutant MalE-254 (Fig. 2a). We previously reported that MalE-254 does not acquire a closed conformation in the presence of maltose, whereas MalE-DW closes rapidly to capture maltose with an affinity ∼60-fold higher than the wild type (
      • Bao H.
      • Duong F.
      Discovery of an auto-regulation mechanism for the maltose transporter MalFGK2.
      ). Together, these results show that open-state MalE, but not closed-liganded MalE, stimulates cleavage of ATP.
      Figure thumbnail gr2
      FIGURE 2Open-state MalE stimulates cleavage of ATP. a and b, hydrolysis of ATP by MalFGK2 under single (a) and multiple (b) turnover conditions. Upper panel shows representative TLC analysis showing ATP hydrolysis by MalFGK2. Lower panel shows the quantification of TLC data from three independent replicates (mean ± S.D.). c and d, retention of ATP hydrolysis products. MalFGK2 was incubated with MalE and maltose in the presence of [α-32P]ATP (c) or [γ-32P]ATP (d) for 5 min under multiple turnover conditions. To detect the fraction of ADP and Pi bound to the transporter, half of the reaction was treated with 50 mm EDTA and analyzed by TLC and autoradiography. The other half was subjected to centrifugal gel filtration on Sepharose G25 before analysis by TLC and autoradiography. Control experiments using the SecA ATPase were performed in parallel (
      • Telmer P.G.
      • Shilton B.H.
      Insights into the conformational equilibria of maltose-binding protein by analysis of high affinity mutants.
      ).
      Under multiple turnover conditions, the amount of ATP hydrolyzed is increased with maltose when compared with MalE alone (Fig. 2b). Because MalE promotes the cleavage of ATP and because maltose inhibits cleavage under single turnover conditions, we reasoned that under these conditions, maltose must accelerate a post-hydrolysis step, such as the release of ADP and/or Pi. As a preliminary assay of whether the release of ADP or Pi is rate-limiting, we determined the amount of ADP and Pi that co-purifies with MalFGK2 (Fig. 2, c and d). A control experiment using the SecA ATPase confirms that SecA, which has a stronger affinity for ADP than Pi (
      • Robson A.
      • Gold V.A.M.
      • Hodson S.
      • Clarke A.R.
      • Collinson I.
      Energy transduction in protein transport and the ATP hydrolytic cycle of SecA.
      ,
      • Sianidis G.
      • Karamanou S.
      • Vrontou E.
      • Boulias K.
      • Repanas K.
      • Kyrpides N.
      • Politou A.S.
      • Economou A.
      Cross-talk between catalytic and regulatory elements in a DEAD motor domain is essential for SecA function.
      ), preferentially co-purifies with ADP (Fig. 2c). In contrast, MalFGK2 seems to preferentially co-purify with Pi (Fig. 2d).

      Release of Pi Limits the Kinetics of the ATPase Cycle

      To determine the step that is limited during the ATP hydrolytic cycle, we analyzed nucleotide turnover in steady state and pre-steady state conditions. The rate of ATP cleavage was determined by rapid mixing, quenched-flow experiments (Fig. 3a). Two distinct kinetic phases were detected in the presence of MalE and maltose: an initial burst of ATP cleavage followed by a slower linear phase at steady state. The rate constant for the initial burst is ∼70 s−1, which is ∼9-fold faster than the steady state rate (7.8 s−1; Table 2). Thus, in the presence of MalE and maltose, the ATPase cycle is evidently limited by a step occurring downstream of ATP hydrolysis. When MalE was omitted from the reaction, the pre-steady and steady state rates were decreased by ∼10-fold, in agreement with the observation that MalE stimulates the ATP cleavage reaction.
      Figure thumbnail gr3
      FIGURE 3Pre-steady state analysis of ATP hydrolysis by MalFGK2. a, the kinetics of ATP cleavage by MalFGK2 (FGK) in the presence or absence of MalE and maltose were determined using quenching-flow rapid mixing. Experiments were carried out using 25 μm MalFGK2 nanodiscs, 100 μm MalE, 1 mm maltose, and 1 mm ATP in ATPase buffer. At the indicated time point, reactions were quenched by buffer A (50 mm Tris-HCl, pH 7.5; 50 mm EDTA). The amount of ATP was quantified with Kinase-Glo reagent. The data were fitted to and summarized in (mean ± S.D., n = 3 independent replicates). b, the kinetics of ADP release from MalFGK2 in the presence or absence of MalE and maltose were determined by stopped-flow rapid mixing. Experiments were carried out with 5 μm MalFGK2 nanodiscs, 50 μm MalE, 1 mm maltose, and 1 mm ATP in ATPase buffer. Release of ADP was measured using the pyruvate kinase/lactate dehydrogenase enzyme-linked assay. The data were fitted to and summarized in (mean ± S.D., n = 3 independent replicates). c, the kinetics of Pi release from MalFGK2 in the presence or absence of MalE and maltose were determined by stopped-flow rapid mixing. Experiments were performed as in b. Release of Pi was measured by using the EnzChek kit. The data were fitted to a simple linear regression model and summarized in (mean ± S.D., n = 3 independent replicates).
      TABLE 2Kinetic parameters of MalFGK2 ATPase
      KcatKburst[ATP]Kburst[ADP]
      s−1s−1s−1
      MalFGK20.3 ± 0.1NANA
      MalFGK2, MalE1.2 ± 0.260 ± 1069 ± 2
      MalFGK2, MalE, maltose7.8 ± 0.370 ± 1566 ± 1
      MalF50027.5 ± 0.490 ± 2068 ± 1
      MalFGK2, MalE, maltose, EIIAGlc1.8 ± 0.24 ± 25.4 ± 0.3
      We then determined the rate of ADP release by stopped-flow kinetics (Fig. 3b). In the presence of MalE and maltose, two distinct kinetic phases were also detected: a fast burst (∼66 s−1) followed by a slower steady state rate (7.2 s−1). Thus, in the presence of MalE and maltose, the ATPase activity of the transporter is not limited by the release of ADP. We next determined the rate of Pi release using the same stopped-flow kinetics (Fig. 3c). In contrast to ATP cleavage and ADP release kinetics, there was no burst for Pi production. Instead, the rate of Pi release was quasi-linear, measured to be 7.5 s−1. We conclude that, under transport conditions, release of Pi limits the ATP hydrolytic cycle, and this step is accelerated by maltose.

      MalF500 Hydrolyzes ATP Constitutively

      Earlier analysis showed that ATP hydrolysis of MalF500 (MalFG338R/N505I) is uncoupled from maltose transport: the mutant hydrolyzes a large amount of ATP in a constitutive manner (
      • Covitz K.M.Y.
      • Panagiotidis C.H.
      • Hor L.I.
      • Reyes M.
      • Treptow N.A.
      • Shuman H.A.
      Mutations that alter the transmembrane signaling pathway in an ATP binding cassette (ABC) transporter.
      ). We therefore determined the ATP hydrolysis kinetics of MalF500 in rapid mixing quenched-flow experiments (Fig. 4). The rate of ATP cleavage in pre-steady states conditions is 90 s−1, a value that is similar to that of MalFGK2 in the presence of MalE (Table 2). The rate of Pi release, which corresponds to the rate of ATP hydrolysis in steady state conditions, is also greatly enhanced (∼3-fold) when compared with the wild type transporter in the presence of MalE and maltose (Table 2). Thus, MalF500 exhibits high rates of ATP cleavage and high rates of Pi release. As a consequence, maltose is unable to stimulate ATP hydrolysis, which results in poor coupling efficiency (
      • Bao H.
      • Dalal K.
      • Wang V.
      • Rouiller I.
      • Duong F.
      The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.
      ).
      Figure thumbnail gr4
      FIGURE 4Pre-steady state analysis of ATP hydrolysis by mutant MalF500. a–c, the kinetics of ATP cleavage (a), ADP release (b), and Pi release (c) were determined as in . The data were fitted to and and summarized in (mean ± S.D., n = 3 independent replicates). FGK, MalFGK2; F500, MalF500.

      The Regulator EIIAGlc Inhibits the Cleavage of ATP

      The transient kinetic analysis above allowed us to examine how the regulatory protein EIIAGlc inhibits the ATPase activity of MalFGK2, a process termed inducer exclusion (
      • Postma P.W.
      • Lengeler J.W.
      • Jacobson G.R.
      Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria.
      ). Recent biochemical and structural data indicate that EIIAGlc binds to the inward-facing MalFGK2 (
      • Bao H.
      • Duong F.
      Phosphatidylglycerol directs binding and inhibitory action of EIIAGlc protein on the maltose transporter.
      ,
      • Chen S.
      • Oldham M.L.
      • Davidson A.L.
      • Chen J.
      Carbon catabolite repression of the maltose transporter revealed by x-ray crystallography.
      ). We therefore tested how EIIAGlc affects the ATP hydrolytic cycle. The results presented in Fig. 5a show that EIIAGlc decreases by ∼20-fold the rate of ATP cleavage in pre-steady conditions and by ∼4-fold in steady state conditions. We conclude that EIIAGlc inhibits the capacity of MalE-MalFGK2 to cleave ATP. As a consequence, EIIAGlc also decreases the rates of ADP release and Pi release from MalFGK2 (Fig. 5, b and c).
      Figure thumbnail gr5
      FIGURE 5EIIAGlc inhibits cleavage of ATP by MalFGK2. Effect of EIIAGlc on MalFGK2 ATPase activity was determined. a–c, the kinetics of ATP cleavage (a), ADP release (b), and Pi release (c) were determined as in . The data (mean ± S.D., n = 3 independent replicates) were fitted to and and summarized in .

      Discussion

      The MalFGK2 transporter is a pioneering model for mechanistic studies on ABC importers; important findings have been derived from its structural and functional analysis (
      • Bordignon E.
      • Grote M.
      • Schneider E.
      The maltose ATP-binding cassette transporter in the 21st century: towards a structural dynamic perspective on its mode of action.
      ,
      • Oldham M.L.
      • Davidson A.L.
      • Chen J.
      Structural insights into ABC transporter mechanism.
      ). In particular, earlier studies showed that MalE and maltose are necessary together to stimulate its ATP hydrolysis rate. However, how the ATPase cycle is accelerated was not determined. Here, we dissected the ATPase reaction into three steps (binding, hydrolysis, and product release) to determine the individual contribution of MalE and maltose, and therefore how substrate translocation is coupled to ATP consumption.
      We show first that binding of ATP to the MalFGK2 does not depend on MalE or maltose. This result was expected because the nucleotide-binding pockets are readily accessible to ATP in the resting transporter (
      • Khare D.
      • Oldham M.L.
      • Orelle C.
      • Davidson A.L.
      • Chen J.
      Alternating access in maltose transporter mediated by rigid-body rotations.
      ). Notably, ATP is sufficient to provoke the dimerization of MalK and the conversion of MalFG to the outward-facing conformation, at least in a transient manner (
      • Daus M.L.
      • Landmesser H.
      • Schlosser A.
      • Müller P.
      • Herrmann A.
      • Schneider E.
      ATP induces conformational changes of periplasmic loop regions of the maltose ATP-binding cassette transporter.
      ,
      • Daus M.L.
      • Grote M.
      • Müller P.
      • Doebber M.
      • Herrmann A.
      • Steinhoff H.J.
      • Dassa E.
      • Schneider E.
      ATP-driven MalK dimer closure and reopening and conformational changes of the “EAA” motifs are crucial for function of the maltose ATP-binding cassette transporter (MalFGK2).
      ,
      • Bao H.
      • Duong F.
      ATP alone triggers the outward facing conformation of the maltose ATP-binding cassette transporter.
      ). This ATP-dependent closure of the NBDs accounts for the basal ATPase activity of the transporter (∼10 nmol/min/mg). However, without MalE and maltose around, the ATP turnover cycle remains inhibited. Indeed, we show that cleavage of ATP absolutely requires MalE. Importantly, we demonstrate using two different mutants that ATP cleavage is stimulated by open-state MalE, and not closed-state MalE (Fig. 2). We reported earlier that the affinity of open-state MalE for outward-facing MalFGK2 is ∼60 nm, whereas the affinity of closed-state MalE for inward-facing MalFGK2 is very low (>100 μm) (
      • Bao H.
      • Duong F.
      Discovery of an auto-regulation mechanism for the maltose transporter MalFGK2.
      ,
      • Bao H.
      • Duong F.
      ATP alone triggers the outward facing conformation of the maltose ATP-binding cassette transporter.
      ). Because the concentration of ATP in cells is >1 mm, the transporter must therefore largely exist in its outward-facing conformation, which has high affinity for open-state MalE. The stability of the complex between open-state MalE and outward-facing MalFGK2 might in turn stabilize the NBDs in a conformation prone to hydrolyze ATP.
      Although MalE accelerates ATP cleavage, we find that the hydrolytic cycle does not proceed forward efficiently. The rate-limiting step corresponds to the release of Pi, and this step is accelerated ∼6-fold in the presence of maltose. Without maltose, the stability of the complex between open-state MalE and outward-facing MalFGK2 might prevent the opening of the MalK dimer, and therefore the release of the ATP hydrolysis products. Interestingly, structural analysis shows that the γ-phosphate makes multiple hydrogen bonds with Walker A and Q-loop, as well as switches histidine from one NBD and the LSGGQ motif from the second NBD (
      • Oldham M.L.
      • Chen J.
      Snapshots of the maltose transporter during ATP hydrolysis.
      ). This high degree of coordination might decrease the dissociation rate of Pi when compared with ADP. At any rate, our current data clearly show that the Kcat value of ATP hydrolysis approaches the Kcleave value when maltose is present, indicating that the steady state complex MalE-MalFGK2 switches from a mostly Pi-bound state to one that rapidly interconverts to the ATP-bound state. We therefore propose that maltose acts like a nucleotide exchange factor to accelerate the replacement of Pi with a new ATP molecule. Importantly, because the release of the hydrolysis products is concomitant with the opening of the MalK dimer and therefore the return of the TMDs to the inward-facing state (
      • Bao H.
      • Duong F.
      Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation.
      ,
      • Khare D.
      • Oldham M.L.
      • Orelle C.
      • Davidson A.L.
      • Chen J.
      Alternating access in maltose transporter mediated by rigid-body rotations.
      ,
      • Lu G.
      • Westbrooks J.M.
      • Davidson A.L.
      • Chen J.
      ATP hydrolysis is required to reset the ATP-binding cassette dimer into the resting-state conformation.
      ,
      • Orelle C.
      • Ayvaz T.
      • Everly R.M.
      • Klug C.S.
      • Davidson A.L.
      Both maltose-binding protein and ATP are required for nucleotide-binding domain closure in the intact maltose ABC transporter.
      ), it is logical to propose that maltose stimulates these conformational changes. Finally, we demonstrate that EIIAGlc inhibits the cleavage of ATP. This last result is in agreement with structural and cross-link data that concluded that EIIAGlc prevents the closure of the NBDs (
      • Bao H.
      • Duong F.
      Phosphatidylglycerol directs binding and inhibitory action of EIIAGlc protein on the maltose transporter.
      ,
      • Chen S.
      • Oldham M.L.
      • Davidson A.L.
      • Chen J.
      Carbon catabolite repression of the maltose transporter revealed by x-ray crystallography.
      ).
      Together, the data allow us to present a transport model (Fig. 6). We reported earlier that MalFGK2 is converted from inward-facing to outward-facing upon binding of ATP (step i and step ii) (
      • Bao H.
      • Duong F.
      ATP alone triggers the outward facing conformation of the maltose ATP-binding cassette transporter.
      ). We also showed that this outward-facing transporter binds open-state MalE with a high degree of affinity (
      • Bao H.
      • Duong F.
      Discovery of an auto-regulation mechanism for the maltose transporter MalFGK2.
      ). Here, we show that binding of open-state MalE induces cleavage of the nucleotide into ADP and Pi (primed state, step iii). Without maltose around, the transporter remains in this conformation; this assembly is stable and can readily be crystallized (
      • Oldham M.L.
      • Khare D.
      • Quiocho F.A.
      • Davidson A.L.
      • Chen J.
      Crystal structure of a catalytic intermediate of the maltose transporter.
      ). With maltose around, the transporter rapidly returns to its initial inward-facing conformation (step iv). Maltose therefore accelerates the release of Pi and thereby the ATP turnover cycle (step v). How maltose enters the transporter, how it is released from open-state MalE, and how it triggers the return of MalFG to the inward-facing state remain to be determined; different models are possible (
      • Chen J.
      Molecular mechanism of the Escherichia coli maltose transporter.
      ,
      • Bao H.
      • Duong F.
      Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation.
      ,
      • Bao H.
      • Duong F.
      Discovery of an auto-regulation mechanism for the maltose transporter MalFGK2.
      ,
      • Shilton B.H.
      The dynamics of the MBP-MalFGK2 interaction: a prototype for binding protein dependent ABC-transporter systems.
      ,
      • Grote M.
      • Polyhach Y.
      • Jeschke G.
      • Steinhoff H.J.
      • Schneider E.
      • Bordignon E.
      Transmembrane signaling in the maltose ABC transporter MalFGK2-E: periplasmic MalF-P2 loop communicates substrate availability to the ATP-bound MalK dimer.
      ,
      • Jacso T.
      • Schneider E.
      • Rupp B.
      • Reif B.
      Substrate transport activation is mediated through second periplasmic loop of transmembrane protein MalF in maltose transport complex of Escherichia coli.
      ,
      • Böhm S.
      • Licht A.
      • Wuttge S.
      • Schneider E.
      • Bordignon E.
      Conformational plasticity of the type I maltose ABC importer.
      ). In our current model (Fig. 6), maltose-bound open-state MalE forms a high-efficiency complex with MalFGK2. This might explain why the affinity of maltose to MalE (∼2–4 μm) is very similar to the Km of the complex MalE-MalFGK2 for maltose (∼2 μm). In addition, the sequential action of MalE and maltose that we report here explains why the ATPase activity of MalFGK2 depends on MalE and why it is coupled to maltose. The importance of this sequential action is best illustrated with the mutant MalF500: cleavage of ATP occurs in the absence of MalE, and release of Pi does not depend on maltose. It follows that the ATPase cycle is uncoupled from transport (
      • Bao H.
      • Dalal K.
      • Wang V.
      • Rouiller I.
      • Duong F.
      The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.
      ). The mutation G338R in MalF500 is located near the periplasmic gate. The destabilization of this region might account for the spontaneous cycle of ATP-driven conversion of the transporter.
      Figure thumbnail gr6
      FIGURE 6Model for the stimulation of MalFGK2 by MalE and maltose. a, in the absence of MalE and maltose, MalFGK2 exhibits low ATP cleavage rate. b, binding of open-state MalE (whether it is bound to maltose or not) stimulates the ATP cleavage rate, but the release of phosphate limits the ATP turnover. c, the presence of maltose alleviates this limitation by accelerating phosphate release. The MalFGK2 ATPase activity is therefore fully stimulated and coupled to transport. Maltose alone is unable to trigger the cleavage of ATP, and therefore translocation is strictly dependent on MalE. The arrow labeled slow indicates the rate-limiting step in each reaction.
      The model in Fig. 6 contrasts with the classic model that assumes that only closed-liganded MalE binds the transporter to trigger the cleavage of ATP. This earlier model bears similarity to that proposed for ABC exporters, i.e. binding of the ligand promotes the closure of the NBDs. Interestingly, in the case of the lipid exporter MsbA, the rate-limiting step corresponds to ATP cleavage, whereas for the multidrug resistance protein 4 (ABCC4), it corresponds to ADP release (
      • Syberg F.
      • Suveyzdis Y.
      • Kötting C.
      • Gerwert K.
      • Hofmann E.
      Time-resolved Fourier transform infrared spectroscopy of the nucleotide-binding domain from the ATP-binding Cassette transporter MsbA: ATP hydrolysis is the rate-limiting step in the catalytic cycle.
      ,
      • Sauna Z.E.
      • Nandigama K.
      • Ambudkar S.V.
      Multidrug resistance protein 4 (ABCC4)-mediated ATP hydrolysis: effect of transport substrates and characterization of the post-hydrolysis transition state.
      ). However, the ATPase rates in MsbA and ABCC4 are only marginally stimulated by the substrate (
      • Syberg F.
      • Suveyzdis Y.
      • Kötting C.
      • Gerwert K.
      • Hofmann E.
      Time-resolved Fourier transform infrared spectroscopy of the nucleotide-binding domain from the ATP-binding Cassette transporter MsbA: ATP hydrolysis is the rate-limiting step in the catalytic cycle.
      ,
      • Doshi R.
      • Ali A.
      • Shi W.
      • Freeman E.V.
      • Fagg L.A.
      • van Veen H.W.
      Molecular disruption of the power stroke in the ATP-binding cassette transport protein MsbA.
      ), similar to the mutant MalF500 in which substrate transport is largely uncoupled to ATP hydrolysis. Thus, although ABC transporters share similar architecture and overall mechanism, the basal ATPase activity that characterizes each transporter might determine the substrate dependence and coupling efficiency, and even perhaps the direction of transport (
      • Grossmann N.
      • Vakkasoglu A.S.
      • Hulpke S.
      • Abele R.
      • Gaudet R.
      • Tampé R.
      Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter.
      ). An approach that systematically determines the ATPase kinetic parameters as developed here would be useful to address how substrate stimulates translocation in other ABC importers.

      Author Contributions

      H. B., K. D., and F. D. conceived the study. H. B. and K. D. designed, performed and analyzed the experiments. H. B., K. D., E. C., and F. G. wrote the paper. All authors analyzed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Dr. Lindsay Eltis for access to the SX.18MV stopped-flow apparatus, and members of our laboratory for critical reading of the manuscript.

      References

        • Higgins C.F.
        ABC transporters: from microorganisms to man.
        Annu. Rev. Cell Biol. 1992; 8: 67-113
        • Cole S.P.
        Multidrug resistance protein 1 (MRP1, ABCC1), a “multitasking” ATP-binding cassette (ABC) transporter.
        J. Biol. Chem. 2014; 289: 30880-30888
        • Cutting G.R.
        Cystic fibrosis genetics: from molecular understanding to clinical application.
        Nat. Rev. Genet. 2015; 16: 45-56
        • Seyffer F.
        • Tampé R.
        ABC transporters in adaptive immunity.
        Biochim. Biophys. Acta. 2015; 1850: 449-460
        • Procko E.
        • O'Mara M.L.
        • Bennett W.F.D.
        • Tieleman D.P.
        • Gaudet R.
        The mechanism of ABC transporters: general lessons from structural and functional studies of an antigenic peptide transporter.
        FASEB J. 2009; 23: 1287-1302
        • Locher K.P.
        Review. Structure and mechanism of ATP-binding cassette transporters.
        Philos. Trans. R. Soc. Lond. B Biol. Sci. 2009; 364: 239-245
        • Chen J.
        Molecular mechanism of the Escherichia coli maltose transporter.
        Curr. Opin. Struct. Biol. 2013; 23: 492-498
        • ter Beek J.
        • Guskov A.
        • Slotboom D.J.
        Structural diversity of ABC transporters.
        J. Gen. Physiol. 2014; 143: 419-435
        • Davidson A.L.
        • Shuman H.A.
        • Nikaido H.
        Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins.
        Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 2360-2364
        • Ames G.F.
        • Liu C.E.
        • Joshi A.K.
        • Nikaido K.
        Liganded and unliganded receptors interact with equal affinity with the membrane complex of periplasmic permeases, a subfamily of traffic ATPases.
        J. Biol. Chem. 1996; 271: 14264-14270
        • Landmesser H.
        • Stein A.
        • Blüschke B.
        • Brinkmann M.
        • Hunke S.
        • Schneider E.
        Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium.
        Biochim. Biophys. Acta. 2002; 1565: 64-72
        • Patzlaff J.S.
        • van der Heide T.
        • Poolman B.
        The ATP/substrate stoichiometry of the ATP-binding cassette (ABC) transporter OpuA.
        J. Biol. Chem. 2003; 278: 29546-29551
        • Borths E.L.
        • Poolman B.
        • Hvorup R.N.
        • Locher K.P.
        • Rees D.C.
        In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake.
        Biochemistry. 2005; 44: 16301-16309
        • Eckford P.D.
        • Sharom F.J.
        Functional characterization of Escherichia coli MsbA: interaction with nucleotides and substrates.
        J. Biol. Chem. 2008; 283: 12840-12850
        • Doshi R.
        • van Veen H.W.
        Substrate binding stabilizes a pre-translocation intermediate in the ATP-binding cassette transport protein MsbA.
        J. Biol. Chem. 2013; 288: 21638-21647
        • Ward A.
        • Reyes C.L.
        • Yu J.
        • Roth C.B.
        • Chang G.
        Flexibility in the ABC transporter MsbA: alternating access with a twist.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 19005-19010
        • Jones P.M.
        • O'Mara M.L.
        • George A.M.
        ABC transporters: a riddle wrapped in a mystery inside an enigma.
        Trends Biochem. Sci. 2009; 34: 520-531
        • Choudhury H.G.
        • Tong Z.
        • Mathavan I.
        • Li Y.
        • Iwata S.
        • Zirah S.
        • Rebuffat S.
        • van Veen H.W.
        • Beis K.
        Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state.
        Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 9145-9150
        • Lee J.Y.
        • Yang J.G.
        • Zhitnitsky D.
        • Lewinson O.
        • Rees D.C.
        Structural basis for heavy metal detoxification by an Atm1-type ABC exporter.
        Science. 2014; 343: 1133-1136
        • Oldham M.L.
        • Chen J.
        Crystal structure of the maltose transporter in a pretranslocation intermediate state.
        Science. 2011; 332: 1202-1205
        • Bao H.
        • Dalal K.
        • Wang V.
        • Rouiller I.
        • Duong F.
        The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.
        Biochim. Biophys. Acta. 2013; 1828: 1723-1730
        • Bao H.
        • Duong F.
        Phosphatidylglycerol directs binding and inhibitory action of EIIAGlc protein on the maltose transporter.
        J. Biol. Chem. 2013; 288: 23666-23674
        • Bao H.
        • Duong F.
        Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation.
        J. Biol. Chem. 2014; 289: 9844-9851
        • Robson A.
        • Gold V.A.M.
        • Hodson S.
        • Clarke A.R.
        • Collinson I.
        Energy transduction in protein transport and the ATP hydrolytic cycle of SecA.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 5111-5116
        • Poolman B.
        • Doeven M.K.
        • Geertsma E.R.
        • Biemans-Oldehinkel E.
        • Konings W.N.
        • Rees D.C.
        Functional analysis of detergent-solubilized and membrane-reconstituted ATP-binding cassette transporters.
        Methods Enzymol. 2005; 400: 429-459
        • Telmer P.G.
        • Shilton B.H.
        Insights into the conformational equilibria of maltose-binding protein by analysis of high affinity mutants.
        J. Biol. Chem. 2003; 278: 34555-34567
        • Gould A.D.
        • Telmer P.G.
        • Shilton B.H.
        Stimulation of the maltose transporter ATPase by unliganded maltose binding protein.
        Biochemistry. 2009; 48: 8051-8061
        • Bao H.
        • Duong F.
        Discovery of an auto-regulation mechanism for the maltose transporter MalFGK2.
        PLoS One. 2012; 7e34836
        • Sianidis G.
        • Karamanou S.
        • Vrontou E.
        • Boulias K.
        • Repanas K.
        • Kyrpides N.
        • Politou A.S.
        • Economou A.
        Cross-talk between catalytic and regulatory elements in a DEAD motor domain is essential for SecA function.
        EMBO J. 2001; 20: 961-970
        • Covitz K.M.Y.
        • Panagiotidis C.H.
        • Hor L.I.
        • Reyes M.
        • Treptow N.A.
        • Shuman H.A.
        Mutations that alter the transmembrane signaling pathway in an ATP binding cassette (ABC) transporter.
        EMBO J. 1994; 13: 1752-1759
        • Postma P.W.
        • Lengeler J.W.
        • Jacobson G.R.
        Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria.
        Microbiol. Rev. 1993; 57: 543-594
        • Chen S.
        • Oldham M.L.
        • Davidson A.L.
        • Chen J.
        Carbon catabolite repression of the maltose transporter revealed by x-ray crystallography.
        Nature. 2013; 499: 364-368
        • Bordignon E.
        • Grote M.
        • Schneider E.
        The maltose ATP-binding cassette transporter in the 21st century: towards a structural dynamic perspective on its mode of action.
        Mol. Microbiol. 2010; 77: 1354-1366
        • Oldham M.L.
        • Davidson A.L.
        • Chen J.
        Structural insights into ABC transporter mechanism.
        Curr. Opin. Struct. Biol. 2008; 18: 726-733
        • Khare D.
        • Oldham M.L.
        • Orelle C.
        • Davidson A.L.
        • Chen J.
        Alternating access in maltose transporter mediated by rigid-body rotations.
        Mol. Cell. 2009; 33: 528-536
        • Daus M.L.
        • Landmesser H.
        • Schlosser A.
        • Müller P.
        • Herrmann A.
        • Schneider E.
        ATP induces conformational changes of periplasmic loop regions of the maltose ATP-binding cassette transporter.
        J. Biol. Chem. 2006; 281: 3856-3865
        • Daus M.L.
        • Grote M.
        • Müller P.
        • Doebber M.
        • Herrmann A.
        • Steinhoff H.J.
        • Dassa E.
        • Schneider E.
        ATP-driven MalK dimer closure and reopening and conformational changes of the “EAA” motifs are crucial for function of the maltose ATP-binding cassette transporter (MalFGK2).
        J. Biol. Chem. 2007; 282: 22387-22396
        • Bao H.
        • Duong F.
        ATP alone triggers the outward facing conformation of the maltose ATP-binding cassette transporter.
        J. Biol. Chem. 2013; 288: 3439-3448
        • Oldham M.L.
        • Chen J.
        Snapshots of the maltose transporter during ATP hydrolysis.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 15152-15156
        • Lu G.
        • Westbrooks J.M.
        • Davidson A.L.
        • Chen J.
        ATP hydrolysis is required to reset the ATP-binding cassette dimer into the resting-state conformation.
        Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 17969-17974
        • Orelle C.
        • Ayvaz T.
        • Everly R.M.
        • Klug C.S.
        • Davidson A.L.
        Both maltose-binding protein and ATP are required for nucleotide-binding domain closure in the intact maltose ABC transporter.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12837-12842
        • Oldham M.L.
        • Khare D.
        • Quiocho F.A.
        • Davidson A.L.
        • Chen J.
        Crystal structure of a catalytic intermediate of the maltose transporter.
        Nature. 2007; 450: 515-521
        • Shilton B.H.
        The dynamics of the MBP-MalFGK2 interaction: a prototype for binding protein dependent ABC-transporter systems.
        Biochim. Biophys. Acta. 2008; 1778: 1772-1780
        • Grote M.
        • Polyhach Y.
        • Jeschke G.
        • Steinhoff H.J.
        • Schneider E.
        • Bordignon E.
        Transmembrane signaling in the maltose ABC transporter MalFGK2-E: periplasmic MalF-P2 loop communicates substrate availability to the ATP-bound MalK dimer.
        J. Biol. Chem. 2009; 284: 17521-17526
        • Jacso T.
        • Schneider E.
        • Rupp B.
        • Reif B.
        Substrate transport activation is mediated through second periplasmic loop of transmembrane protein MalF in maltose transport complex of Escherichia coli.
        J. Biol. Chem. 2012; 287: 17040-17049
        • Böhm S.
        • Licht A.
        • Wuttge S.
        • Schneider E.
        • Bordignon E.
        Conformational plasticity of the type I maltose ABC importer.
        Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 5492-5497
        • Syberg F.
        • Suveyzdis Y.
        • Kötting C.
        • Gerwert K.
        • Hofmann E.
        Time-resolved Fourier transform infrared spectroscopy of the nucleotide-binding domain from the ATP-binding Cassette transporter MsbA: ATP hydrolysis is the rate-limiting step in the catalytic cycle.
        J. Biol. Chem. 2012; 287: 23923-23931
        • Sauna Z.E.
        • Nandigama K.
        • Ambudkar S.V.
        Multidrug resistance protein 4 (ABCC4)-mediated ATP hydrolysis: effect of transport substrates and characterization of the post-hydrolysis transition state.
        J. Biol. Chem. 2004; 279: 48855-48864
        • Doshi R.
        • Ali A.
        • Shi W.
        • Freeman E.V.
        • Fagg L.A.
        • van Veen H.W.
        Molecular disruption of the power stroke in the ATP-binding cassette transport protein MsbA.
        J. Biol. Chem. 2013; 288: 6801-6813
        • Grossmann N.
        • Vakkasoglu A.S.
        • Hulpke S.
        • Abele R.
        • Gaudet R.
        • Tampé R.
        Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter.
        Nat, Commun. 2014; 55419