Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Conformational dynamics of ligand-dependent alternating access in LeuT

Nature Structural & Molecular Biology volume 21, pages 472–479 (2014)Cite this article

Subjects

Abstract

The leucine transporter (LeuT) from Aquifex aeolicus is a bacterial homolog of neurotransmitter/sodium symporters (NSSs) that catalyze reuptake of neurotransmitters at the synapse. Crystal structures of wild-type and mutants of LeuT have been interpreted as conformational states in the coupled transport cycle. However, the mechanistic identities inferred from these structures have not been validated, and the ligand-dependent conformational equilibrium of LeuT has not been defined. Here, we used distance measurements between spin-label pairs to elucidate Na+- and leucine-dependent conformational changes on the intracellular and extracellular sides of the transporter. The results identify structural motifs that underlie the isomerization of LeuT between outward-facing, inward-facing and occluded states. The conformational changes reported here present a dynamic picture of the alternating-access mechanism of LeuT and NSSs that is different from the inferences reached from currently available structural models.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Model of LeuT alternating access inferred from the crystal structures.
Figure 2: Na+-induced opening and Na+- and leucine-induced closing of the LeuT extracellular side.
Figure 3: Fluctuation dynamics of TMs 6 and 7 and the N-terminal segment mediate the opening of the intracellular side of LeuT.
Figure 4: β-OG stabilizes the outward-facing conformation of LeuT in the presence of Na+ and leucine.
Figure 5: The Y268A or R5A mutations induce structural rearrangements in LeuT.
Figure 6: Models of LeuT conformational changes derived from restrained ensemble simulations.
Figure 7: Cartoon model of LeuT transport derived from EPR data.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Rudnick, G. in Neurotransmitter Transporters (ed. Reith, M.A.) 25–52 (Humana Press, 2002).

  2. Iversen, L. Neurotransmitter transporters and their impact on the development of psychopharmacology. Br. J. Pharmacol. 147 (suppl. 1), S82–S88 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Amara, S.G. & Sonders, M.S. Neurotransmitter transporters as molecular targets for addictive drugs. Drug Alcohol Depend. 51, 87–96 (1998).

    CAS  PubMed  Google Scholar 

  4. Singh, S.K. & Leu, T. A prokaryotic stepping stone on the way to a eukaryotic neurotransmitter transporter structure. Channels (Austin) 2, 380–389 (2008).

    Google Scholar 

  5. Gether, U., Andersen, P.H., Larsson, O.M. & Schousboe, A. Neurotransmitter transporters: molecular function of important drug targets. Trends Pharmacol. Sci. 27, 375–383 (2006).

    CAS  PubMed  Google Scholar 

  6. Yamashita, A., Singh, S.K., Kawate, T., Jin, Y. & Gouaux, E. Crystal structure of a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporters. Nature 437, 215–223 (2005).

    CAS  PubMed  Google Scholar 

  7. Singh, S.K., Piscitelli, C.L., Yamashita, A. & Gouaux, E. A competitive inhibitor traps LeuT in an open-to-out conformation. Science 322, 1655–1661 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Beuming, T., Shi, L., Javitch, J.A. & Weinstein, H. A comprehensive structure-based alignment of prokaryotic and eukaryotic neurotransmitter/Na+ symporters (NSS) aids in the use of the LeuT structure to probe NSS structure and function. Mol. Pharmacol. 70, 1630–1642 (2006).

    CAS  PubMed  Google Scholar 

  9. Kanner, B.I. & Zomot, E. Sodium-coupled neurotransmitter transporters. Chem. Rev. 108, 1654–1668 (2008).

    CAS  PubMed  Google Scholar 

  10. Kristensen, A.S. et al. SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol. Rev. 63, 585–640 (2011).

    CAS  PubMed  Google Scholar 

  11. Forrest, L.R. et al. Mechanism for alternating access in neurotransmitter transporters. Proc. Natl. Acad. Sci. USA 105, 10338–10343 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Mitchell, P. A general theory of membrane transport from studies of bacteria. Nature 180, 134–136 (1957).

    CAS  PubMed  Google Scholar 

  13. Jardetzky, O. Simple allosteric model for membrane pumps. Nature 211, 969–970 (1966).

    CAS  PubMed  Google Scholar 

  14. Vidaver, G.A. Inhibition of parallel flux and augmentation of counter flux shown by transport models not involving a mobile carrier. J. Theor. Biol. 10, 301–306 (1966).

    CAS  PubMed  Google Scholar 

  15. Patlak, C.S. Contributions to the theory of active transport: II. The gate type non-carrier mechanism and generalizations concerning tracer flow, efficiency, and measurement of energy expenditure. Bull. Math. Biophys. 19, 209–235 (1957).

    Google Scholar 

  16. Krishnamurthy, H. & Gouaux, E. X-ray structures of LeuT in substrate-free outward-open and apo inward-open states. Nature 481, 469–474 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Shimamura, T. et al. Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1. Science 328, 470–473 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Watanabe, A. et al. The mechanism of sodium and substrate release from the binding pocket of vSGLT. Nature 468, 988–991 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Perez, C., Koshy, C., Yildiz, O. & Ziegler, C. Alternating-access mechanism in conformationally asymmetric trimers of the betaine transporter BetP. Nature 490, 126–130 (2012).

    CAS  PubMed  Google Scholar 

  20. Shaffer, P.L., Goehring, A., Shankaranarayanan, A. & Gouaux, E. Structure and mechanism of a Na+-independent amino acid transporter. Science 325, 1010–1014 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Shi, L., Quick, M., Zhao, Y., Weinstein, H. & Javitch, J.A. The mechanism of a neurotransmitter:sodium symporter–inward release of Na+ and substrate is triggered by substrate in a second binding site. Mol. Cell 30, 667–677 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Shi, L. & Weinstein, H. Conformational rearrangements to the intracellular open states of the LeuT and ApcT transporters are modulated by common mechanisms. Biophys. J. 99, L103–L105 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Forrest, L.R. & Rudnick, G. The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters. Physiology (Bethesda) 24, 377–386 (2009).

    CAS  Google Scholar 

  24. McHaourab, H.S., Steed, P.R. & Kazmier, K. Toward the fourth dimension of membrane protein structure: insight into dynamics from spin-labeling EPR spectroscopy. Structure 19, 1549–1561 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Freed, D.M., Horanyi, P.S., Wiener, M.C. & Cafiso, D.S. Conformational exchange in a membrane transport protein is altered in protein crystals. Biophys. J. 99, 1604–1610 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Cross, T.A., Sharma, M., Yi, M. & Zhou, H.X. Influence of solubilizing environments on membrane protein structures. Trends Biochem. Sci. 36, 117–125 (2011).

    CAS  PubMed  Google Scholar 

  27. Hubbell, W.L., McHaourab, H.S., Altenbach, C. & Lietzow, M.A. Watching proteins move using site-directed spin labeling. Structure 4, 779–783 (1996).

    CAS  PubMed  Google Scholar 

  28. Jeschke, G. & Polyhach, Y. Distance measurements on spin-labelled biomacromolecules by pulsed electron paramagnetic resonance. Phys. Chem. Chem. Phys. 9, 1895–1910 (2007).

    CAS  PubMed  Google Scholar 

  29. Zhao, Y. et al. Single-molecule dynamics of gating in a neurotransmitter transporter homologue. Nature 465, 188–193 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Claxton, D.P. et al. Ion/substrate-dependent conformational dynamics of a bacterial homolog of neurotransmitter:sodium symporters. Nat. Struct. Mol. Biol. 17, 822–829 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Kniazeff, J. et al. An intracellular interaction network regulates conformational transitions in the dopamine transporter. J. Biol. Chem. 283, 17691–17701 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Georgieva, E.R., Borbat, P.P., Ginter, C., Freed, J.H. & Boudker, O. Conformational ensemble of the sodium-coupled aspartate transporter. Nat. Struct. Mol. Biol. 20, 215–221 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Hänelt, I., Wunnicke, D., Bordignon, E., Steinhoff, H.J. & Slotboom, D.J. Conformational heterogeneity of the aspartate transporter GltPh . Nat. Struct. Mol. Biol. 20, 210–214 (2013).

    PubMed  Google Scholar 

  34. Polyhach, Y., Bordignon, E. & Jeschke, G. Rotamer libraries of spin labelled cysteines for protein studies. Phys. Chem. Chem. Phys. 13, 2356–2366 (2011).

    CAS  PubMed  Google Scholar 

  35. Polyhach, Y., Godt, A., Bauer, C. & Jeschke, G. Spin pair geometry revealed by high-field DEER in the presence of conformational distributions. J. Magn. Reson. 185, 118–129 (2007).

    CAS  PubMed  Google Scholar 

  36. Quick, M. et al. Binding of an octylglucoside detergent molecule in the second substrate (S2) site of LeuT establishes an inhibitor-bound conformation. Proc. Natl. Acad. Sci. USA 106, 5563–5568 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Islam, S.M., Stein, R.A., McHaourab, H.S. & Roux, B. Structural refinement from restrained-ensemble simulations based on EPR/DEER data: application to T4 lysozyme. J. Phys. Chem. B 117, 4740–4754 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Roux, B. & Islam, S.M. Restrained-ensemble molecular dynamics simulations based on distance histograms from double electron-electron resonance spectroscopy. J. Phys. Chem. B 117, 4733–4739 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Zhao, Y. et al. Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue. Nature 474, 109–113 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Quick, M. & Javitch, J.A. Monitoring the function of membrane transport proteins in detergent-solubilized form. Proc. Natl. Acad. Sci. USA 104, 3603–3608 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Pannier, M., Veit, S., Godt, A., Jeschke, G. & Spiess, H.W. Dead-time free measurement of dipole-dipole interactions between electron spins. J. Magn. Reson. 142, 331–340 (2000).

    CAS  PubMed  Google Scholar 

  42. Jeschke, G. DEER distance measurements on proteins. Annu. Rev. Phys. Chem. 63, 419–446 (2012).

    CAS  PubMed  Google Scholar 

  43. Jeschke, G. et al. DeerAnalysis2006: a comprehensive software package for analyzing pulsed ELDOR data. Appl. Magn. Reson. 30, 473–498 (2006).

    CAS  Google Scholar 

  44. Chiang, Y.W., Borbat, P.P. & Freed, J.H. The determination of pair distance distributions by pulsed ESR using Tikhonov regularization. J. Magn. Reson. 172, 279–295 (2005).

    CAS  PubMed  Google Scholar 

  45. Brooks, B.R. et al. CHARMM: The biomolecular simulation program. J. Comput. Chem. 30, 1545–1614 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. MacKerell, A.D. et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998).

    CAS  PubMed  Google Scholar 

  47. Mackerell, A.D., Feig, M. & Brooks, C.L. Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J. Comput. Chem. 25, 1400–1415 (2004).

    CAS  PubMed  Google Scholar 

  48. Lazaridis, T. Effective energy function for proteins in lipid membranes. Proteins 52, 176–192 (2003).

    CAS  PubMed  Google Scholar 

  49. Lazaridis, T. & Karplus, M. Effective energy function for proteins in solution. Proteins 35, 133–152 (1999).

    CAS  PubMed  Google Scholar 

  50. Jo, S., Kim, T., Iyer, V.G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008).

    CAS  PubMed  Google Scholar 

  51. Adelman, S.A. & Doll, J.D. Generalized Langevin equation approach for atom-solid-surface scattering - general formulation for classical scattering off harmonic solids. J. Chem. Phys. 64, 2375–2388 (1976).

    CAS  Google Scholar 

Download references

Acknowledgements

The authors gratefully acknowledge R. Stein for assistance with EPR data collection and EPR distance analysis. We thank H. Koteiche and P.R. Steed for critical reading and editing of the manuscript. We thank Extreme Science and Engineering Discovery Environment (XSEDE) for computer time. This work was supported by US National Institutes of Health grants U54-GM087519 (H.S.M., S.S., H.W., J.A.J., S.M.I. and B.R.), K05DA022414 (J.A.J.) and P01DA012408 (H.W.). K.K. was supported by a predoctoral National Research Service Award (F31-MH095383-01).

Author information

Author notes

  1. Kelli Kazmier and Shruti Sharma: These authors contributed equally to this work.

Authors and Affiliations

  1. Chemical and Physical Biology Program, Vanderbilt University, Nashville, Tennessee, USA

    Kelli Kazmier & Hassane S Mchaourab

  2. Department of Molecular Physiology and Biophysics, Nashville, Tennessee, USA

    Shruti Sharma & Hassane S Mchaourab

  3. Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Matthias Quick & Jonathan A Javitch

  4. Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Matthias Quick & Jonathan A Javitch

  5. Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, USA

    Matthias Quick & Jonathan A Javitch

  6. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA

    Shahidul M Islam & Benoît Roux

  7. Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York, USA

    Harel Weinstein

  8. HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, New York, USA

    Harel Weinstein

  9. Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Jonathan A Javitch

Authors
  1. Kelli Kazmier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shruti Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthias Quick
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shahidul M Islam
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Benoît Roux
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Harel Weinstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jonathan A Javitch
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hassane S Mchaourab
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.K. and S.S. constructed mutants, expressed and purified protein, prepared samples and conducted DEER experiments. K.K. and H.S.M. designed the DEER experiments. K.K., S.S. and H.S.M. analyzed DEER data and interpreted the distance distributions. M.Q. designed, conducted and analyzed leucine-binding and alanine-transport experiments. S.M.I. and B.R. refined structural models with the restrained ensemble simulations method. All authors contributed to the mechanistic interpretation of the data, wrote and edited the manuscript.

Corresponding author

Correspondence to Hassane S Mchaourab.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Na+-dependent leucine binding to spin-labeled LeuT variants.

Leu binding in the presence of Na+ to spin-labeled LeuT was determined by means of the scintillation proximity assay (SPA) as described in the online Methods. Data were normalized to the level of leucine binding to the LeuT-WT, determined concurrently (n ≥ 3). Data are shown as the mean ± S.E.M. (a) Binding of 1.5 μM 3H-Leu (9 Ci/mmol) to 50 ng of spin-labeled LeuT in the presence of 150 mM NaCl. Most mutants with the exception of those in TM6 or in the Y268A or R5A backgrounds bind Leu with similar affinity and stoichiometry as the WT. (b) Saturation binding of 3H-Leu (9 Ci/mmol) by select mutants as indicated. Saturation binding for the tested LeuT variants was normalized with regard to the level of binding observed for LeuT-WT at 5 μM final concentration. The right shift of the curve indicates that these mutants, destabilized either by the Y268A or R5A substitutions or spin labeling of TM6, have lower affinity to leucine. Nevertheless, they bind leucine to the same level as WT at high leucine concentrations.

Supplementary Figure 2 Na+-dependent [3H]alanine uptake by spin-labeled LeuT variants.

Uptake of 3H-Ala by spin-labeled LeuT mutants reconstituted into proteoliposomes was conducted as described in the online Methods. Data were normalized to the level of alanine transport by the LeuT-WT, determined concurrently (n ≥ 3). Data are shown as the mean ± S.E.M. (a) Alanine transport (1 μM final concentration; 20 Ci/mmol) was monitored for 5 and 30 minutes, as indicated. (b) Kinetics of 3H-Ala transport. The initial rates of 3H-Ala transport were measured for 1 minute time periods at 3H-Ala concentrations ranging from 0.125 – 5 μM (with specific radioactivites from 49.5 Ci/mmol – 5 Ci/mmol). The transport rates of most mutants do not differ significantly from WT. For F208C-F306C, the deviation is attributable to a change in Km.

Supplementary Figure 3 Spin labeling of TM6 residues.

(a) The intracellular network of hydrogen bonding and electrostatic interactions stabilized by Y268A of IL3. (b) The network of spin label pairs monitoring the movement of TM6b and the loop connecting to TM7a is shown by black spheres connected by solid lines. b) Na+- and Leu-dependent changes in the distance distributions demonstrating the movement of TM6b in the WT (solid lines) and the R5A (dashed lines) backgrounds. The broad distributions hinder analysis of the magnitude of distance changes but are consistent with the ligand-dependent equilibrium of this TM between multiple conformations.

Supplementary Figure 4 Crystal structures of LeuT underestimate the closing of the extracellular vestibule.

Comparison of experimental distance distributions (solid lines) with predicted distributions based on the “inward-facing” (3TT3), “outward-facing” (3TT1) and “substrate-occluded” (2A65) crystal structures. The MMM package was used to generate predicted distance distributions on the extracellular side. This comparison demonstrates that the experimental distances in the Na+- and Leu-bound state fall outside the predicted distributions regardless of the crystal structure. Because MMM typically overestimates the distribution width, we interpret the systematic deviations between calculated and experimental distributions as evidence that the substrate-occluded conformation we observe in solution is not represented in the crystallographic record. Hence the crystal structures underestimate the closing of the extracellular side upon Na+ and Leu binding. The only exception to this seems to be EL4, for which the predicated distribution from the inward-facing structure overlaps the experimental distribution obtained in the presence of Na+ and Leu.

Supplementary Figure 5 The ‘substrate-occluded’ crystal structure overestimates the closing of TMs 1a and 2 on the intracellular side.

Comparison between experimental and MMM-predicted distances shows systematic deviations in the distributions of TMs 1a and 2. In contrast to the extracellular side, here the average distances are larger than those predicted by the occluded structure.

Supplementary Figure 6 Comparison of distance distributions at selected sites in the WT (solid line) and the Y268A and R5A (dashed lines) backgrounds.

The Y268A mutation induces movement of TMs 1a and 5 in the direction expected based on the inward-facing crystal structure. The (*) indicates components arising from aggregation during concentration of the mutants after gel filtration. The addition of Na+ and Leu does not reset the distributions back to WT-like. This suggests a loss of conformational coupling. Areas known to be a part of the rigid C structure (grey helices) also show changes in the distance distributions in the Y268A background. We interpret these changes as indicative of the global destabilization effects of these mutations.

Supplementary Figure 7 The LeuT scaffold.

(a) The static scaffold of LeuT. Narrow distance distributions between TMs 2, 3, 4, 5, 8, and 9 suggest a predominantly rigid scaffold. TM1a may undergo small scale movement as indicated by the width change in the TM1a-TM9 distribution. (b) Evidence of small scale movements of TMs 10 and 11 relative to TM2. This movement appears to be coordinated with that of TM7a as their pairwise distributions do not show Na+-dependent or Na+- and Leu-dependent changes in average distance or width.

Supplementary Figure 8 Analysis of DEER distance distributions.

(a) in WT background, (b) in WT background in the presence of β-OG, and (c) in R5A and Y268A backgrounds. For each mutant, we present the background-corrected decays and fits (left), the L curves with α parameter selected at the elbow (middle), and the normalized distance distributions (right).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 15175 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kazmier, K., Sharma, S., Quick, M. et al. Conformational dynamics of ligand-dependent alternating access in LeuT. Nat Struct Mol Biol 21, 472–479 (2014). https://doi.org/10.1038/nsmb.2816

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2816

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing