Article | Published:

The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism

Nature Structural & Molecular Biology volume 23, pages 256263 (2016) | Download Citation

Abstract

Secondary transporters use alternating-access mechanisms to couple uphill substrate movement to downhill ion flux. Most known transporters use a 'rocking bundle' motion, wherein the protein moves around an immobile substrate-binding site. However, the glutamate-transporter homolog GltPh translocates its substrate-binding site vertically across the membrane, through an 'elevator' mechanism. Here, we used the 'repeat swap' approach to computationally predict the outward-facing state of the Na+/succinate transporter VcINDY, from Vibrio cholerae. Our model predicts a substantial elevator-like movement of VcINDY's substrate-binding site, with a vertical translation of ~15 Å and a rotation of ~43°. Our observation that multiple disulfide cross-links completely inhibit transport provides experimental confirmation of the model and demonstrates that such movement is essential. In contrast, cross-links across the VcINDY dimer interface preserve transport, thus revealing an absence of large-scale coupling between protomers.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Protein Data Bank

References

  1. 1.

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

  2. 2.

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

  3. 3.

    , , , & Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 445, 387–393 (2007).

  4. 4.

    , & Unlocking the molecular secrets of sodium-coupled transporters. Nature 459, 347–355 (2009).

  5. 5.

    , , , & Crystal structure of a bacterial homologue of Na+/Cl-dependent neurotransmitter transporters. Nature 437, 215–223 (2005).

  6. 6.

    , , , & The major facilitator superfamily (MFS) revisited. FEBS J. 279, 2022–2035 (2012).

  7. 7.

    & Evolutionary relationship between 5+5 and 7+7 inverted repeat folds within the amino acid-polyamine-organocation superfamily. Proteins 82, 336–346 (2014).

  8. 8.

    et al. Gating topology of the proton-coupled oligopeptide symporters. Structure 23, 290–301 (2015).

  9. 9.

    , , , & Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1. Proc. Natl. Acad. Sci. USA 111, 14752–14757 (2014).

  10. 10.

    et al. Conformational dynamics of ligand-dependent alternating access in LeuT. Nat. Struct. Mol. Biol. 21, 472–479 (2014).

  11. 11.

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

  12. 12.

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

  13. 13.

    , , & Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats. Proc. Natl. Acad. Sci. USA 106, 20752–20757 (2009).

  14. 14.

    , & Transport mechanism of a bacterial homologue of glutamate transporters. Nature 462, 880–885 (2009).

  15. 15.

    et al. A two-domain elevator mechanism for sodium/proton antiport. Nature 501, 573–577 (2013).

  16. 16.

    , , & SLC13 family of Na+-coupled di- and tri-carboxylate/sulfate transporters. Mol. Aspects Med. 34, 299–312 (2013).

  17. 17.

    , , & Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae. J. Gen. Physiol. 143, 745–759 (2014).

  18. 18.

    , & TCDB: the Transporter Classification Database for membrane transport protein analyses and information. Nucleic Acids Res. 34, D181–D186 (2006).

  19. 19.

    , , & Structure and mechanism of a bacterial sodium-dependent dicarboxylate transporter. Nature 491, 622–626 (2012).

  20. 20.

    et al. Phylogenetic characterization of transport protein superfamilies: superiority of SuperfamilyTree programs over those based on multiple alignments. J. Mol. Microbiol. Biotechnol. 21, 83–96 (2011).

  21. 21.

    , , & The ion transporter superfamily. Biochim. Biophys. Acta 1618, 79–92 (2003).

  22. 22.

    , , , & Family resemblances: a common fold for some dimeric ion-coupled secondary transporters. J. Gen. Physiol. 146, 423–434 (2015).

  23. 23.

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

  24. 24.

    & The alternating-access mechanism of MFS transporters arises from inverted-topology repeats. J. Mol. Biol. 407, 698–715 (2011).

  25. 25.

    et al. A model-structure of a periplasm-facing state of the NhaA antiporter suggests the molecular underpinnings of pH-induced conformational changes. J. Biol. Chem. 287, 18249–18261 (2012).

  26. 26.

    , , & Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms. Front. Pharmacol. 6, 183 (2015).

  27. 27.

    , , & Conformational changes required for H+/Cl exchange mediated by a CLC transporter. Nat. Struct. Mol. Biol. 21, 456–463 (2014).

  28. 28.

    & Rigidity of the subunit interfaces of the trimeric glutamate transporter GltT during translocation. J. Mol. Biol. 372, 565–570 (2007).

  29. 29.

    , & On the accuracy of homology modeling and sequence alignment methods applied to membrane proteins. Biophys. J. 91, 508–517 (2006).

  30. 30.

    , , & Relation between sequence and structure in membrane proteins. Bioinformatics 29, 1589–1592 (2013).

  31. 31.

    , , & Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature 431, 811–818 (2004).

  32. 32.

    , , & Mechanism of Na+-dependent citrate transport from the structure of an asymmetrical CitS dimer. eLife 4, e09375 (2015).

  33. 33.

    , , , & Structure and transport mechanism of the sodium/proton antiporter MjNhaP1. eLife 3, e03583 (2014).

  34. 34.

    , & Structure and substrate ion binding in the sodium/proton antiporter PaNhaP. eLife 3, e03579 (2014).

  35. 35.

    , & Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4 Å. Nature 483, 489–493 (2012).

  36. 36.

    et al. Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters. eLife 3, e03604 (2014).

  37. 37.

    et al. Structural fold and binding sites of the human Na+-phosphate cotransporter NaPi-II. Biophys. J. 106, 1268–1279 (2014).

  38. 38.

    et al. Crystal structure of the Alcanivorax borkumensis YdaH transporter reveals an unusual topology. Nat. Commun. 6, 6874 (2015).

  39. 39.

    et al. Structure and function of Neisseria gonorrhoeae MtrF illuminates a class of antimetabolite efflux pumps. Cell Rep. 11, 61–70 (2015).

  40. 40.

    , & Structure based identification of inhibitors for the SLC13 family of Na+/dicarboxylate cotransporters. Biochemistry 54, 4900–4908 (2015).

  41. 41.

    , , & Interactions of benzylpenicillin and non-steroidal anti-inflammatory drugs with the sodium-dependent dicarboxylate transporter NaDC-3. Cell. Physiol. Biochem. 14, 415–424 (2004).

  42. 42.

    & Nonsteroidal anti-inflammatory drugs and other anthranilic acids inhibit the Na+/dicarboxylate symporter from Staphylococcus aureus. Biochemistry 52, 2924–2932 (2013).

  43. 43.

    & TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res. 33, 2302–2309 (2005).

  44. 44.

    , , , & SymD webserver: a platform for detecting internally symmetric protein structures. Nucleic Acids Res. 42, W296–W300 (2014).

  45. 45.

    Quality measures for protein alignment benchmarks. Nucleic Acids Res. 38, 2145–2153 (2010).

  46. 46.

    & Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637 (1983).

  47. 47.

    , , , & ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529–W533 (2010).

  48. 48.

    & Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

  49. 49.

    , & Model quality assessment for membrane proteins. Bioinformatics 26, 3067–3074 (2010).

  50. 50.

    , , & Procheck: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993).

  51. 51.

    , , & OPM: orientations of proteins in membranes database. Bioinformatics 22, 623–625 (2006).

  52. 52.

    , , , & The PMDB Protein Model Database. Nucleic Acids Res. 34, D306–D309 (2006).

  53. 53.

    et al. The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins. J. Struct. Funct. Genomics 11, 191–199 (2010).

  54. 54.

    et al. The substrate-binding protein imposes directionality on an electrochemical sodium gradient-driven TRAP transporter. Proc. Natl. Acad. Sci. USA 106, 1778–1783 (2009).

Download references

Acknowledgements

We thank A. Banerjee for helpful discussions and M. Maduke, J. Faraldo-Gómez, G. Rudnick and M. Mayer for critical review of the manuscript. A.V.-J. is supported as a recipient of the L'Oreal Chile–United Nations Educational, Scientific and Cultural Organization (UNESCO) Women in Science Fellowship and the L'Oreal-UNESCO Rising Talent Award. This work was supported by the Division of Intramural Research of the US National Institutes of Health, National Institute of Neurological Disorders and Stroke.

Author information

Author notes

    • Gabriel A Fitzgerald
    •  & Desirée Kaufmann

    Present addresses: Department of Physiology, Weill Cornell Medical College, New York, New York, USA (G.A.F.), and Institute of Molecular Biology, Mainz, Germany (D.K.).

Affiliations

  1. Membrane Transport Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA.

    • Christopher Mulligan
    • , Gabriel A Fitzgerald
    •  & Joseph A Mindell
  2. Computational Structural Biology Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA.

    • Cristina Fenollar-Ferrer
    • , Ariela Vergara-Jaque
    •  & Lucy R Forrest
  3. Max Planck Institute of Biophysics, Frankfurt am Main, Germany.

    • Cristina Fenollar-Ferrer
    •  & Desirée Kaufmann
  4. Protein/Peptide Sequencing Facility, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA.

    • Yan Li

Authors

  1. Search for Christopher Mulligan in:

  2. Search for Cristina Fenollar-Ferrer in:

  3. Search for Gabriel A Fitzgerald in:

  4. Search for Ariela Vergara-Jaque in:

  5. Search for Desirée Kaufmann in:

  6. Search for Yan Li in:

  7. Search for Lucy R Forrest in:

  8. Search for Joseph A Mindell in:

Contributions

L.R.F. and J.A.M. conceived the project. C.F.-F., A.V.-J. and D.K. carried out computational modeling, and C.F.-F. and L.R.F. analyzed data and directed computational modeling efforts. J.A.M. and C.M. designed and planned experiments. C.M. performed experiments and supervised G.A.F., who performed dimer-interface experiments. Y.L. performed MS and interpreted MS data. C.M., C.F.-F., L.R.F. and J.A.M. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Lucy R Forrest or Joseph A Mindell.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Table 1

  2. 2.

    Supplementary Data Set 1

    Raw gel images from all figures

Text files

  1. 1.

    Supplementary Data Set 2

    Model of VcINDY in the outward-facing conformation

Videos

  1. 1.

    Predicted transport-associated conformational change in VcINDY

    The conformational change predicted for VcINDY is modeled by morphing the VcINDY inward-facing X- ray structure (PDB ID: 4F35) (start) with the repeat-swapped model of the outwardfacing conformation (end point). The interpolation between structure and model was done using the Morph2 server (http://molmovdb.mbb.yale.edu/molmovdb/morph/). VcINDY is represented as cartoon helices and the coloring scheme reflects the division between scaffold (blues), oligomerization domain (dark blue) and transport domain (orange), as shown in Figure 1. The sodium ion and the citrate are shown as purple and yellow spheres, respectively. The protein is viewed from the plane of the membrane, first, from the perspective of the other protomer, and secondly, along the dimer axis.

  2. 2.

    Cross-linking during the transport-associated conformational change in VcINDY

    See legend to Supplementary Movie 1 for more detail. Here, the Cβ atom of each of the residue pairs that crosslink in the outwardfacing conformation is also shown as a sphere, color-coded by crosslinking pair as follows: Ala120 and Val165 in magenta, Thr154 and Val272 in green and Ala346 and Val364 in red.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb.3166

Further reading