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Substrate-modulated gating dynamics in a

LETTER

doi:10.1038/nature09971

Substrate-modulated gating dynamics in a Na 1-coupled neurotransmitter transporter homologue

Yongfang Zhao 1,2,4*,Daniel S.Terry 5*,Lei Shi 5,6*,Matthias Quick 1,2,4,Harel Weinstein 5,6,Scott C.Blanchard 5&Jonathan A.Javitch 1,2,3,4

Neurotransmitter/Na 1symporters (NSSs)terminate neuronal sig-nalling by recapturing neurotransmitter released into the synapse in a co-transport (symport)mechanism driven by the Na 1electro-chemical gradient 1–6.NSSs for dopamine,noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine 1,as well as by antidepressants 7.The crystal structure of LeuT,a prokaryotic NSS homologue,revealed an occluded con-formation in which a leucine (Leu)and two Na 1are bound deep within the protein 8.This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold 9–22.Subsequently,an ‘outward-open’conformation was determined in the presence of the inhibitor tryptophan 23,and the Na 1-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy 24.In addition,single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation,which involve the movement of transmem-brane helix TM1a away from the transmembrane helical bundle 22.We investigated how substrate binding is coupled to structural transitions in LeuT during Na 1-coupled transport.Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein.In the presence of alanine,a substrate that is transported 10-fold faster than leucine 15,25,we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency.Collectively,our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2)binding site within the extra-cellular vestibule 15,20.Substrate binding in this S2site appears to act cooperatively with the primary substrate (S1)binding site to control

intracellular gating more than 30A

?away,in a manner that allows the Na 1

gradient to power the transport mechanism.

The experiments were performed on LeuT engineered to contain a 15-amino-acid,carboxy-terminal biotinylation domain 26and site-specifically labelled with the fluorophores Cy3and Cy5maleimide at residue position 7,after replacing the native His residue with Cys (H7C)in the amino-terminal loop close to TM1,and at position 86(R86C)in intracellular loop (IL)1(Methods).Direct observations of conformational processes within the intracellular gate region of LeuT (Supplementary Fig.1)were made using a wide field imaging strategy employing prism-based total internal reflection (Methods,Fig.1a).As described 22,fluorescence resonance energy transfer (FRET)imaging of

LeuT revealed two readily distinguished states (FRET efficiency ,0.51and ,0.75)in the presence of 200mM K 1and the nominal absence of Na 1(Fig.1b),consistent with the existence of two distinct conforma-tions of the intracellular gate that differ by ,13A

?in the distance separating the fluorophore pair.

In experiments imaging LeuT dynamics with increasing Na 1con-centrations,Hidden Markov Modelling revealed that the distribution of low-and high-FRET conformations of LeuT was altered by Na 1with an effector concentration for half-maximum response (EC 50)of 10.9mM (Fig.1b,c),consistent with the EC 50for Na 1-dependent stimulation of substrate binding and transport 15.Na 1decreased the overall frequency of transitions (Fig.1d,e)through the preferential stabilization (,7-fold)of the inward-closed state.During the direct imaging of individual LeuT molecules (Fig.1f),slow,spontaneous tran-sitions between open and closed states,initially observed in 200mM K 1,were dramatically decreased on exchange into Na 1-containing buffer,leading to the preferential stabilization of the inward-closed state.

Reasoning that substrate-induced intracellular gating might be observed best under conditions mimicking the relatively low intracel-lular Na 1,we performed experiments at Na 1concentrations sufficient for Leu binding but below the EC 50of Na 1.However,even at 2mM Na 1,Leu shifted the population towards the closed intracellular gate conformation (Supplementary Fig.2a,b)through a ,3.5-fold stabiliza-tion of this state (Supplementary Fig.2c).These effects,which result in a global decrease in transition frequency (Supplementary Fig.2d),were recapitulated at the level of individual LeuT molecules (Supplementary Fig.2e).Thus,while unambiguously demonstrating binding of both Na 1and Leu to LeuT,these results corroborate our earlier finding that Leu binding has the net effect of diminishing the likelihood of intracel-lular gate opening.One possible explanation for these observations is that Leu’s high affinity for the transporter 15makes it a poor substrate for transport,which in our measurements is manifested in the greatly extended lifetime of the closed state.To test this hypothesis,intracellular gate dynamics were assessed in the presence of the more efficiently transported substrate Ala.

In stark contrast to Leu,under otherwise identical conditions,increas-ing Ala concentrations did not shift the FRET distribution towards the closed state (Fig.2a,b).Instead,a strong,Ala-concentration-dependent enhancement of transition rates was observed.In 2mM Na 1,Ala enhanced the transition rates between inward-open and inward-closed statesbyasmuchas ,4-fold(Fig.2c,d).Thisresultwasdirectlyconfirmed at the scale of individual molecules on exchange into Ala-containing buffer (Fig.2e).Similar enhancements in transition frequency were also

*These authors contributed equally to this work.

Center for Molecular Recognition,Columbia University College of Physicians and Surgeons,630West 168th Street,New York,New York 10032,USA.2Department of Psychiatry,Columbia University College of Physicians and Surgeons,630West 168th Street,New York,New York 10032,USA.3Department of Pharmacology,Columbia University College of Physicians and Surgeons,630West 168th Street,New York,New York 10032,USA.4Division of Molecular Therapeutics,New York State Psychiatric Institute,1051Riverside Drive,New York,New York 10032,USA.5Department of Physiology and Biophysics,Weill Medical College of Cornell University,1300York Avenue,New York,New York 10021,USA.6HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine,Weill Cornell Medical College,Cornell University,1300York Avenue,New York,New York 10021,USA.2J U L Y 2011|V O L 474|N A T U R E |109

observed for H7C/T515C-LeuT (Supplementary Fig.3).In accordance with such effects,which required both Na 1and Ala,the lifetimes of the inward-open or inward-closed FRET states were not significantly affected by Ala alone (in the nominal absence of Na 1);at 250m M Ala,the transition frequency increased in a Na 1-concentration-dependent fashion (Supplementary Fig.4).

Using transition state theory (Methods),we found that the intracel-lular open and closed FRET states of LeuT were separated by a large activation barrier (D G {<80kJ mol 21).Ala does not alter the relative occupancies of open and closed states,but instead lowers the activation barrier for both open-to-closed and closed-to-open transitions by approximately 3kJ mol 21(about the energy of a hydrogen bond).By contrast,Leu raised the activation barrier for the closed-to-open trans-ition by as much as 4kJ mol 21,apparently through ground-state sta-bilization of the closed state.

Hypothesizing that the observed dynamics reflect Ala’s acceleration of the opening-closing cycles of the intracellular gate required for the transport mechanism,we performed experiments in the presence of the transport inhibitor clomipramine (CMI),a tricyclic antidepressant that is known to bind in an extracellular vestibule above the Na 1and S1binding sites 25,27,28.Many of the residues shown to interact with antidepressants in these structures are also part of the S2site 25,27.As substrate binding in the S2site is thought to allosterically trigger intra-cellular release of Na 1and substrate from the S1site 15(also see Supplementary Fig.1),CMI should block Ala-induced intracellular gating dynamics.Indeed,in the presence of both Na 1and Ala,CMI essentially eliminated intracellular gate opening,stabilizing LeuT in a high-FRET,inward-closed conformation (Supplementary Fig.5a–c).

This observation is consistent with CMI competitively blocking sub-strate binding to the S2site 15,thereby preventing Ala-induced opening and closing of the intracellular gate,and inhibiting transport.This result was again confirmed by direct imaging of individual LeuT mol-ecules in Na 1and Ala-containing buffer on addition of CMI (Fig.2f).The detergent n-octyl-b -D -glucopyranoside also inhibited intracellular gating dynamics (Supplementary Fig.5a–c),consistent with its capa-city to disrupt the Na 1-coupled transport mechanism 20by competing with substrate binding to the S2site 20,23.

To probe whether Ala binding to the S1and/or S2site(s)was responsible for lowering the activation barrier for intracellular gating dynamics,single-molecule FRET experiments were performed in the background of either an F253A or L400S mutation within the S1or S2site,respectively (Fig.3a,Supplementary Fig.1).These mutations disrupt substrate binding to LeuT,decreasing the stoichiometry of substrate:LeuT binding under saturating conditions from 2:1in wild-type LeuT,to 1:1in both mutants (Fig.4a).Mutation of F253blocks substrate binding to the S1site and also abrogates transport (Fig.4a,b;Supplementary Fig.6),while having little or no effect on Na 1binding (Supplementary Table 1).Despite evidence that Ala bound to the S2site in the context of the F253A mutation (Fig.4a),Ala failed to increase intracellular gating dynamics of the mutant protein (Fig.4c).Similarly,despite evidence of Ala binding to the S1site (Fig.4a),no increase in intracellular gating dynamics was observed when the S2site was disrupted by the L400S mutation (Fig.4c).These findings support the notion that substrate occupancy in the S2site is critical for the allosteric mechanism that controls intracellular gate opening and the release of substrate from the S1

0.04.5 × 10–49.0 × 10–4

1.3 × 10–3

0.40.60.8Initial FRET F i n a l F R E T

Initial FRET Initial FRET [NaCl] (mM)

Initial FRET

4812012

456

0.20.40.60.8F l u o r e s c e n c e

Time (min)

FRET

D w e l l t i m e (s )

[NaCl] (mM)

86C

7C BAP

biotin

F R E T

Figure 1|Effect of Na 1on LeuT dynamics.a ,Experimental set-up:H7C/R86C-LeuT labelled with Cy3and Cy5(stars)was immobilized via a biotin acceptor peptide (BAP)on a passivated glass surface and illuminated using total internal reflection.FRET traces (.110per condition)were collected with varying concentrations of Na 1(160-ms time resolution for all,except 30–50mM with 400ms).b ,Histograms of FRET traces,filtered to remove

fluorophore dark states.c ,Fraction of time in the lower-FRET open state (black

open squares)and the high-FRET closed state (red filled circles).d ,Transition density plot:average FRET values before (x axis)and after (y axis)each transition were plotted as a two-dimensional chart in transitions per second (scale at right;Na 1concentrations are indicated).e ,Average dwell times in each state.f ,Representative traces (donor in green,acceptor in red,FRET in blue,and predicted state sequence (idealization)in red),where the solution was exchanged at 2min from K 1to Na 1(200mM).Error bars,s.d.of $100bootstrap samples.

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site 15,and demonstrate that substrate binding to both the S1and S2sites is necessary to trigger intracellular gating.

In order to probe whether Ala binding to the S1and S2sites is also sufficient to promote intracellular gating and transport,experiments were performed in the presence of Li 1in place of Na 1.In the presence of saturating Li 1concentrations (.150mM),we found that Ala binds LeuT with a 2:1stoichiometry consistent with both S1and S2site occupancy (Fig.4a).Li 1,like Na 1,stabilized the inward-closed state (Supplementary Fig.7),but,in the presence of Li 1,Ala failed to accel-erate intracellular gating dynamics and no substrate transport was observed (Fig.4c).Instead,the inward-closed conformation of LeuT

was modestly stabilized in the presence of Ala (,2-fold reduction in the rate of gate opening,k closed-open )(Fig.4c).These data demonstrate that Ala binding to the S1and S2sites in the presence of Li 1does not lower the activation barrier to intracellular gating as observed in the presence of Na 1.

Prompted by these experimental observations,computational studies were performed to investigate how both Na 1and Li 1can support substrate binding to LeuT,whereas only Na 1leads to substrate-induced dynamics of the intracellular gate and to transport.These studies also served to identify both local changes produced in the region of the ion binding sites and critical elements in the allosteric pathway linking the

O c c u p a n c y (%)

Time (min)

Ala 0.20.40.60.8 Time (min)

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a e

CMI d

Initial FRET

F i n a l F R E T Initial FRET

Initial FRET Initial FRET

c log [Ala] (μM)

log [Ala] (μM)

D w e l l t i m e (s )

0.0

4.5 × 10–49.0 × 10–4

1.3 × 10–3

Figure 2|Effect of alanine on LeuT dynamics.Single-molecule FRET traces (.90per condition)were collected at 160-ms time resolution with 2mM Na 1and varying concentrations of Ala.a ,Histograms of FRET data from each condition.Hidden Markov Modelling analysis revealed the fraction of time (b )and average dwell times (c )in the lower-FRET open state (black open squares)and the high-FRET closed state (red filled circles).d ,Transition

density plots as in Fig.1d (Ala concentrations are indicated).e ,f ,Representative FRET traces (blue)with idealization (red)from experiments where solution was exchanged at 2min:e ,2mM Na 1,adding 250m M Ala;f ,2mM Na 1and 250m M Ala,adding the inhibitor clomipramine (CMI;0.5mM).Error bars,s.d.of $100bootstrap samples.

TM10

TM6

E419

F252D404

R30

E290Li1Li2

T254G408-T409

TM6

TM10

Na1Na2Li1Li2

Na1

Na2L400

S2F253

F259 V412-V413L400

F253

Figure 3|The configuration of TM6–TM10interactions induced by Na 1binding cannot be matched by Li 1binding.a ,Representative snapshot taken from the Na-only simulation,showing water molecules (red spheres)

occupying the S1and S2sites (white dotted ellipses).Residues L400in the S2site and F253in the S1site,which were mutated to affect substrate binding,are shown as light green sticks.b ,The different effects that Li 1and Na 1binding

have on the interacting residues of TM6and TM10.The TM6/TM10interface is indicated by the dashed ellipse in magenta.c ,The bulge around G408in TM10,which is present only when Na 1is bound but not when Li 1replaces it.In b and c ,side chains and backbones coloured according to atom types are from the Li-only conformation,while those from the Na-only conformation are rendered in grey.

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substrate binding sites and the intracellular gate https://www.wendangku.net/doc/0f6581164.html,parative analysis of separate molecular dynamics (MD)simulations of LeuT,performed with either Na 1or Li 1occupying the established Na 1bind-ing sites and in the absence of amino acid substrate (termed Na-only 24and Li-only,respectively)revealed significant differences in TM–TM interactions (Fig.3b,c),which are described in detail in Supplementary Information.The Na1/Li1binding site and its neighbouring interaction network,which are crucial for the proper propagation of the allosteric effects from the S2to S1site (see Supplementary Information for details)and onward to the intracellular side to open the transport pathway,are sensitive to the unique combination of the ionic radius of the Na 1cation and the charge redistribution it causes.The structural consequences of the ion-specific effects appear to be propagated through the cluster of aromatic residues at the heart of the S1binding site,and result in dif-ferent configurations of the bulge in the middle of TM10(Fig.3,Sup-plementary Fig.8).

The positions of the structural elements involved in this propagation mechanism make them critical for transmitting conformational changes deeper into the TM bundle towards the intracellular end of the transporter (Fig.3b,c).Such changes include local alterations in the vicinity of E419,a residue known from the crystal structure to interact with E62in TM2,with the backbone of the unwound portion of TM6(proximal to F259of the aromatic cluster and the S1binding site),and two water molecules 29.Reconfiguration of this region,including residue T418,on simulated inward movement of the substrate 15was previously shown to enable the penetration of water from the intracellular side of LeuT as a result of an opening at IL115.The resulting dissociation of IL1from interactions with R5and D369and the destabilization of the network of intracellular interactions detected in the simulations (Supplementary Fig.9)is associated with the observed outward move-ment of TM1a 22that is essential for the simulated release of substrate to the intracellular side.

Owing to the different effects of Li 1and Na 1,Ala binding in both the S1and S2sites in the presence of Li 1would not engender the ordered series of local conformational rearrangements expected in the presence of Na 1.These rearrangements originate in the S2site and need to be propagated as described above through changes in the Na1and S1sites to enable water penetration from the cytoplasmic side of LeuT and the outward movement of TM1a.Their absence when Li 1substitutes for Na 1would explain why substrate-induced acceleration of gating dynamics was not observed experimentally.

Na 1binding,which stabilizes the inward-closed state,does not hasten gate closure but,instead,slightly stabilizes the inward-open state as well,by raising the energy barrier to the conformational transi-tion.In contrast,Ala binding to LeuT in the presence of Na 1shortens

not only the inward-closed,but also the inward-open,lifetime (Fig.2).Thus,bound Ala facilitates both the opening of the intracellular gate and its subsequent closure by reducing the activation barrier for such conformational transitions.One possible explanation for this obser-vation is that binding of substrate in the S2site triggers the opening of the intracellular gate and release of the S1substrate to the cytoplasm.In the absence of S1substrate and bound Na 1,substrate in the S2site may then facilitate intracellular gate closure.It is tempting to speculate that the S2substrate,in the presence of extracellular Na 1,may move to the S1site with high efficiency owing to its very high local concentra-tion,thereby facilitating a subsequent transport cycle.

Collectively,our findings support the notion that the observed movements of TM1a and its environment are associated with LeuT intracellular gating 22in a manner that is directly linked to the Na 1-driven transport mechanism.Thus,results obtained with the slowly transported substrate,Leu,and the relatively rapidly transported sub-strate,Ala,establish a relationship between the rates of intracellular gating and substrate transport.The role of substrate binding at the S2site in the process of allostery and molecular recognition is further highlighted by the comparative effects of CMI and Ala binding to this site in the presence of Na 1.The former stabilizes a closed intracellular gate conformation,whereas the latter substantially lowers the activa-tion barrier to gate opening and thereby allows the energy of the Na 1gradient to drive the transport mechanism.

After the present manuscript had been submitted,a report 30was published that concluded,on the basis of a variety of binding measure-ments,that LeuT has only a single high-affinity substrate site.In con-trast,our substrate binding measurements clearly show a stoichiometry of 2:1,consistent with high affinity binding to both the S1and S2sites 20.Half of this binding is lost in the S2-site mutant that also exhibits a loss of substrate-induced single-molecule dynamics and transport (Fig.4).Although the loss of substrate-induced dynamics and transport in the S2-site mutant could conceivably be explained solely by a long-range allosteric effect of the mutation,all our data to date are most consistent with a two-substrate-site model in which the absence of either S1or S2substrate binding results in a profound attenuation of transporter dynamics and function.We are currently uncovering the reasons for the discrepancy between the data of ref.30and our own data,and will report our findings in due course.

METHODS SUMMARY

LeuT mutants were expressed in Escherichia coli ,purified,and labelled on targeted engineered cysteines with Cy3and Cy5maleimide.The functional properties of the labelled constructs were determined by measuring Leu binding and Na 1by scintillation proximity assay,and Ala transport was measured after reconstitution of the protein into proteoliposomes.Purified,labelled protein was immobilized onto a passivated glass surface via a streptavidin-biotin linkage (shown schematically in Fig.1a).Fluorescence data were acquired using a prism-based total internal reflection (TIR)microscope.FRET efficiency was calculated and analysis of fluor-escence and FRET traces was achieved using semi-automated analysis software developed for this application.The single-molecule traces were analysed for LeuT in the presence and absence of the substrates Na 1,Leu and Ala,and on addition of the transport inhibitors CMI and n-octyl-b -D -glucopyranoside,and in response to mutations of the S1and S2binding sites.MD simulations were carried out with the protein immersed in an all-atom model of the membrane,solvated with water molecules,ions and ligands.Long equilibrations (totalling .2m s)were run to assess conformational changes,with more than one MD trajectory collected for every configuration mentioned.

Full Methods and any associated references are available in the online version of the paper at https://www.wendangku.net/doc/0f6581164.html,/nature.

Received 12August 2010;accepted 25February 2011.

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Supplementary Information is linked to the online version of the paper at

https://www.wendangku.net/doc/0f6581164.html,/nature.

Acknowledgements We thank R.Altman for assistance in preparing reagents for single-molecule experiments and F.Carvalho for the preparation of membranes. Molecular graphics were prepared with https://www.wendangku.net/doc/0f6581164.html,putations were performed on Ranger at the Texas Advanced Computing Center(TG-MCB090022)and the David A.Cofrin computational infrastructure of the Institute for Computational Biomedicine at Weill Cornell Medical College.This work was supported in part by National Institutes of Health grants DA17293and DA022413(J.A.J.),DA12408(H.W.),DA023694(L.S.), the Irma T.Hirschl/Monique Weill-Caulier trusts(S.C.B.)and the Lieber Center for Schizophrenia Research and Treatment.D.S.T.is supported by the Tri-Institutional Training Program in Computational Biology and Medicine.

Author Contributions Y.Z.expressed,purified and labelled the LeuT mutants.M.Q.and Y.Z.performed the functional characterization of the mutants.Y.Z.and D.S.T.designed, carried out and analysed the single-molecule experiments;L.S.and H.W.designed and analysed the computational studies,which were carried out by L.S.;S.C.B.and J.A.J. helped to design the biochemical and single-molecule experiments and,with L.S.and H.W.,helped to interpret the data.All the authors contributed to writing and editing the manuscript.

Author Information Reprints and permissions information is available at

https://www.wendangku.net/doc/0f6581164.html,/reprints.The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at

https://www.wendangku.net/doc/0f6581164.html,/nature.Correspondence and requests for materials should be addressed to J.A.J.(jaj2@https://www.wendangku.net/doc/0f6581164.html,)or S.C.B(scb2005@https://www.wendangku.net/doc/0f6581164.html,).

LETTER RESEARCH

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METHODS

Protein expression and purification.LeuT variants were expressed in E.coli C41(DE3)as described15.For functional studies,LeuT variants were expressed from pQO18or derivatives thereof carrying the indicated mutations20,whereas for single-molecule FRET studies,biotin acceptor peptide-tagged LeuT variants were expressed in pETO18G and its derivatives22.Protein was purified by immobilized metal(Ni21)affinity chromatography using a Ni21Sepharose6FastFlow column (GE Healthcare)22.For fluorescent labelling of LeuT,Cy3-maleimide and Cy5-maleimide(GE Healthcare)were added at an equimolar ratio(200m M total)for 1h while the protein was bound to the Ni21resin22.Free dye was removed before the elution of LeuT with300mM imidazole.

Scintillation proximity-based binding studies.Binding of3H-leucine or3H-alanine(146Ci mmol21and49.4Ci mmol21,respectively;both from Moravek) to purified LeuT-variants was measured with the scintillation proximity assay (SPA)as described15,with25ng of purified protein per assay in buffer composed of150mM Tris/MES,pH7.5/50mM NaCl/1mM TCEP/0.1%n-dodecyl-b-D-maltopyranoside or50mM Tris/MES,pH7.5/150mM LiCl/1mM TCEP/0.1% n-dodecyl-b-D-maltopyranoside.To determine the molar ratio of Leu(or Ala)-to LeuT,binding samples were incubated with increasing concentrations of radioligand and measured in the SPA c.p.m.mode of the Wallac1450MicroBeta counter(Perkin Elmer).The efficiency of detection was calculated with standard curves of known concentrations of3H-Leu or3H-Ala.The standard curves were used to transform c.p.m.into the amount of bound substrate15.The amount of LeuT in the SPA assays was determined31.SPA-based binding studies using2m M[22Na]Cl(1,017mCi mg21; Perkin Elmer)were performed in150–200mM Tris/MES,pH7.5/1mM TCEP/0.1% n-dodecyl-b-D-maltopyranoside in the presence of0–50mM NaCl(equimolar replacement of Tris/MES to maintain a total molarity of200mM)15.All experiments were repeated at least in duplicate with triplicate determination of all individual data points.Kinetic constants(shown6the s.e.m.of the fit)were obtained by fitting the data from independent experiments to global fitting in Prism or SigmaPlot.

3H-Ala transport in proteoliposomes.Proteoliposomes were prepared as described15.The accumulation of3H-Ala(49.4Ci mmol21;Moravek)was mea-sured at23u C in assay buffer composed of150/50mM Tris/MES(pH8.5)and 50mM NaCl/150mM LiCl.The reaction was quenched by the addition of ice-cold assay buffer without radiotracer and the proteoliposomes were collected on GF-75 glass fibre filters(Advantec)before the determination of the accumulated c.p.m.by liquid scintillation counting.

Single-molecule FRET imaging experiments.Fluorescence experiments were performed using a prism-based total internal reflection fluorescence(TIRF)micro-scope as previously described22,32.Microfluidic imaging chambers were passivated with a mixture of PEG and biotin-PEG and incubated with0.8m M streptavidin (Invitrogen).Cy3/Cy5-labelled,biotinylated LeuT molecules were surface immobi-lized through biotin-streptavidin interaction.Cy3fluorophores were excited by the evanescent wave generated by total internal reflection(TIR)of a single-frequency light source(Ventus532,Laser Quanta).Photons emitted from Cy3 and Cy5were collected using a1.2NA603water-immersion objective(Nikon) and optical treatments were used to separate Cy3and Cy5frequencies onto a cooled, back-thinned EMCCD camera(Cascade512,Photometrics).Fluorescence data were acquired using Metamorph(Universal Imaging Corporation).

All experiments were performed in buffer containing50mM Tris/MES,pH7.5, 10%glycerol,0.02%(w/v)DDM,5mM2-mercaptoethanol and200mM salt(KCl or NaCl,as specified).We used an oxygen-scavenging environment(1unit per ml glucose oxidase,8units per ml catalase,0.1%(v/v)glucose)containing2mM cyclooctatetraene in all experiments to minimize photobleaching.

Analysis of single-molecule fluorescence data was performed using custom soft-ware written in MATLAB(MathWorks).A subset of the acquired traces was selected for further analysis using the following criteria:(1)single-step donor photobleaching,(2)signal-to-background noise ratio(SNR)$8,(3),4donor blinking events,(4)non-zero FRET efficiency for at least60s.Additional manual trace selection was performed to refine the data,where selected traces were required to have:(1)stable total fluorescence intensity(I D1I A)and(2)at least one trans-ition between clearly defined FRET states with anti-correlated transitions in donor/ acceptor intensity or a single dwell in a clearly-defined FRET state.We found this process to be effective in removing artefacts and spurious noise without introducing significant bias(see Supplementary Discussion and Supplementary Fig.10). Kinetic analysis was performed to idealize FRET traces and calculate average dwell times using a three-state model as previously described22.Error bars for transition rate estimates and FRET histograms were calculated as the standard deviation of100bootstrap samples of the traces.Error bars for state occupancies were calculated from1,000bootstrap samples.

Transition rates were interpreted using transition state theory,where the open and closed states are considered ground states separated by a large (D G{<80kJ mol21)activation barrier(the transition state).The energy required to achieve the transition state(and cross the barrier)was calculated as:

D G{~{RT ln

hk i,j

k B T

where R is the gas constant,T is absolute temperature(296K),h is Planck’s constant,k is the measured transition rate,from state i to state j,and k B is Boltzmann’s constant.Changes in the activation barrier energy(DD G{)were calculated from the difference in forward and reverse rates observed in the absence and presence of substrate.

Molecular dynamics.The Li1-only simulation was performed on a system pre-pared as described24.Briefly,it consisted of more than77,000atoms,including the explicit membrane model,solvating water molecules and the various ions and ligands.All the Na1ions in the system were replaced with Li1.The parameters for Li1were from ref.33.All MD simulations were carried out with the NAMD program under constant temperature(310K)and constant pressure(1atm)con-ditions.Long equilibration runs were performed to allow the system to transition to a new stable conformation.The inward-closed and inward-open conformations described in Supplementary Fig.9were based on the simulations described previ-ously22.More than one MD trajectory was collected for every configuration studied.Each individual trajectory was at least360ns,and the longest trajectory for each configuration was720ns.All the results reinforced the conclusions,and the structural and dynamic insights described in the main text were revealed as the common features and trends of parallel independent MD runs.

31.Schaffner,W.&Weissmann,C.A rapid,sensitive,and specific method for the

determination of protein in dilute solution.Anal.Biochem.56,502–514(1973).

32.Munro,J.B.,Altman,R.B.,O’Connor,N.&Blanchard,S.C.Identification of two

distinct hybrid state intermediates on the ribosome.Mol.Cell25,505–517(2007).

33.Caplan,D.A.,Subbotina,J.O.&Noskov,S.Y.Molecular mechanism of ion-ion and

ion-substrate coupling in the Na1-dependent leucine transporter LeuT.Biophys.J.

95,4613–4621(2008).

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