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.

Mechanism of extracellular ion exchange and binding-site occlusion in a sodium/calcium exchanger

Abstract

Na+/Ca2+ exchangers use the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+ and play a central role in Ca2+ homeostasis. Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state. This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3:1 Na+/Ca2+-exchange stoichiometry and reveals the conformational changes at the onset of the alternating-access transport mechanism. An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations. These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion occupancy, thereby explaining the emergence of strictly coupled Na+/Ca2+ antiport.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Na+ binding to outward-facing NCX_Mj.
Figure 2: Na+-occupancy-dependent conformational changes in NCX_Mj.
Figure 3: Divalent cation binding and apo structure of NCX_Mj.
Figure 4: Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB 3V5U20) upon sequential displacement of Na+.
Figure 5: Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX.
Figure 6: Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX.
Figure 7: Structural mechanism of extracellular forward ion exchange in NCX.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Blaustein, M.P. & Lederer, W.J. Sodium/calcium exchange: its physiological implications. Physiol. Rev. 79, 763–854 (1999).

    Article  CAS  Google Scholar 

  2. DiPolo, R. & Beaugé, L. Sodium/calcium exchanger: influence of metabolic regulation on ion carrier interactions. Physiol. Rev. 86, 155–203 (2006).

    Article  CAS  Google Scholar 

  3. Clapham, D.E. Calcium signaling. Cell 131, 1047–1058 (2007).

    Article  CAS  Google Scholar 

  4. Berridge, M.J., Bootman, M.D. & Roderick, H.L. Calcium signalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4, 517–529 (2003).

    Article  CAS  Google Scholar 

  5. Hilgemann, D.W. Unitary cardiac Na+, Ca2+ exchange current magnitudes determined from channel-like noise and charge movements of ion transport. Biophys. J. 71, 759–768 (1996).

    Article  CAS  Google Scholar 

  6. Hilgemann, D.W., Nicoll, D.A. & Philipson, K.D. Charge movement during Na+ translocation by native and cloned cardiac Na+/Ca2+ exchanger. Nature 352, 715–718 (1991).

    Article  CAS  Google Scholar 

  7. Reeves, J.P. & Hale, C.C. The stoichiometry of the cardiac sodium-calcium exchange system. J. Biol. Chem. 259, 7733–7739 (1984).

    CAS  PubMed  Google Scholar 

  8. Blaustein, M.P. & Russell, J.M. Sodium-calcium exchange and calcium-calcium exchange in internally dialyzed squid giant axons. J. Membr. Biol. 22, 285–312 (1975).

    Article  CAS  Google Scholar 

  9. Rasgado-Flores, H. & Blaustein, M.P. Na/Ca exchange in barnacle muscle cells has a stoichiometry of 3 Na+/1 Ca2+ . Am. J. Physiol. 252, C499–C504 (1987).

    Article  CAS  Google Scholar 

  10. Kimura, J., Noma, A. & Irisawa, H. Na-Ca exchange current in mammalian heart cells. Nature 319, 596–597 (1986).

    Article  CAS  Google Scholar 

  11. Kang, T.M. & Hilgemann, D.W. Multiple transport modes of the cardiac Na+/Ca2+ exchanger. Nature 427, 544–548 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Hilgemann, D.W. Regulation and deregulation of cardiac Na+-Ca2+ exchange in giant excised sarcolemmal membrane patches. Nature 344, 242–245 (1990).

    Article  CAS  Google Scholar 

  14. Matsuoka, S., Nicoll, D.A., Reilly, R.F., Hilgemann, D.W. & Philipson, K.D. Initial localization of regulatory regions of the cardiac sarcolemmal Na+-Ca2+ exchanger. Proc. Natl. Acad. Sci. USA 90, 3870–3874 (1993).

    Article  CAS  Google Scholar 

  15. Philipson, K.D. & Nicoll, D.A. Sodium-calcium exchange: a molecular perspective. Annu. Rev. Physiol. 62, 111–133 (2000).

    Article  CAS  Google Scholar 

  16. Nicoll, D.A., Hryshko, L.V., Matsuoka, S., Frank, J.S. & Philipson, K.D. Mutation of amino acid residues in the putative transmembrane segments of the cardiac sarcolemmal Na+-Ca2+ exchanger. J. Biol. Chem. 271, 13385–13391 (1996).

    Article  CAS  Google Scholar 

  17. Nicoll, D.A., Ottolia, M. & Philipson, K.D. Toward a topological model of the NCX1 exchanger. Ann. NY Acad. Sci. 976, 11–18 (2002).

    Article  CAS  Google Scholar 

  18. Ren, X., Nicoll, D.A., Xu, L., Qu, Z. & Philipson, K.D. Transmembrane segment packing of the Na+/Ca2+ exchanger investigated with chemical cross-linkers. Biochemistry 49, 8585–8591 (2010).

    Article  CAS  Google Scholar 

  19. Cai, X. & Lytton, J. The cation/Ca2+ exchanger superfamily: phylogenetic analysis and structural implications. Mol. Biol. Evol. 21, 1692–1703 (2004).

    Article  CAS  Google Scholar 

  20. Liao, J. et al. Structural insight into the ion-exchange mechanism of the sodium/calcium exchanger. Science 335, 686–690 (2012).

    Article  CAS  Google Scholar 

  21. Almagor, L. et al. Functional asymmetry of bidirectional Ca2+-movements in an archaeal sodium-calcium exchanger (NCX_Mj). Cell Calcium 56, 276–284 (2014).

    Article  CAS  Google Scholar 

  22. Marinelli, F. et al. Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation. Proc. Natl. Acad. Sci. USA 111, E5354–E5362 (2014).

    Article  CAS  Google Scholar 

  23. Blaustein, M.P. & Santiago, E.M. Effects of internal and external cations and of ATP on sodium-calcium and calcium-calcium exchange in squid axons. Biophys. J. 20, 79–111 (1977).

    Article  CAS  Google Scholar 

  24. Trosper, T.L. & Philipson, K.D. Effects of divalent and trivalent cations on Na+-Ca2+ exchange in cardiac sarcolemmal vesicles. Biochim. Biophys. Acta 731, 63–68 (1983).

    Article  CAS  Google Scholar 

  25. DiPolo, R. & Beaugé, L. Asymmetrical properties of the Na-Ca exchanger in voltage-clamped, internally dialyzed squid axons under symmetrical ionic conditions. J. Gen. Physiol. 95, 819–835 (1990).

    Article  CAS  Google Scholar 

  26. Matsuoka, S. & Hilgemann, D.W. Steady-state and dynamic properties of cardiac sodium-calcium exchange: ion and voltage dependencies of the transport cycle. J. Gen. Physiol. 100, 963–1001 (1992).

    Article  CAS  Google Scholar 

  27. Miura, Y. & Kimura, J. Sodium-calcium exchange current: dependence on internal Ca and Na and competitive binding of external Na and Ca. J. Gen. Physiol. 93, 1129–1145 (1989).

    Article  CAS  Google Scholar 

  28. Waight, A.B. et al. Structural basis for alternating access of a eukaryotic calcium/proton exchanger. Nature 499, 107–110 (2013).

    Article  CAS  Google Scholar 

  29. Nishizawa, T. et al. Structural basis for the counter-transport mechanism of a H+/Ca2+ exchanger. Science 341, 168–172 (2013).

    Article  CAS  Google Scholar 

  30. Wu, M. et al. Crystal structure of Ca2+/H+ antiporter protein YfkE reveals the mechanisms of Ca2+ efflux and its pH regulation. Proc. Natl. Acad. Sci. USA 110, 11367–11372 (2013).

    Article  CAS  Google Scholar 

  31. Giladi, M. et al. Asymmetric preorganization of inverted pair residues in the sodium-calcium exchanger. Sci. Rep. 6, 20753 (2016).

    Article  CAS  Google Scholar 

  32. Iwamoto, T., Uehara, A., Imanaga, I. & Shigekawa, M. The Na+/Ca2+ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca2+ affinity. J. Biol. Chem. 275, 38571–38580 (2000).

    Article  CAS  Google Scholar 

  33. John, S.A., Liao, J., Jiang, Y. & Ottolia, M. The cardiac Na+-Ca2+ exchanger has two cytoplasmic ion permeation pathways. Proc. Natl. Acad. Sci. USA 110, 7500–7505 (2013).

    Article  CAS  Google Scholar 

  34. Ottolia, M., Nicoll, D.A. & Philipson, K.D. Mutational analysis of the alpha-1 repeat of the cardiac Na+-Ca2+ exchanger. J. Biol. Chem. 280, 1061–1069 (2005).

    Article  CAS  Google Scholar 

  35. Reeves, J.P. & Sutko, J.L. Competitive interactions of sodium and calcium with the sodium-calcium exchange system of cardiac sarcolemmal vesicles. J. Biol. Chem. 258, 3178–3182 (1983).

    CAS  PubMed  Google Scholar 

  36. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4, 706–731 (2009).

    Article  CAS  Google Scholar 

  37. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  38. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  39. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  40. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  41. Petrek, M. et al. CAVER: a new tool to explore routes from protein clefts, pockets and cavities. BMC Bioinformatics 7, 316 (2006).

    Article  Google Scholar 

  42. Phillips, J.C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).

    Article  CAS  Google Scholar 

  43. 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).

    Article  CAS  Google Scholar 

  44. Mackerell, A.D. Jr., Feig, M. & Brooks, C.L. III. 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).

    Article  CAS  Google Scholar 

  45. Staritzbichler, R., Anselmi, C., Forrest, L.R. & Faraldo-Gómez, J.D. GRIFFIN: a versatile methodology for optimization of protein-lipid interfaces for membrane protein simulations. J. Chem. Theory Comput. 7, 1167–1176 (2011).

    Article  CAS  Google Scholar 

  46. Piana, S. & Laio, A. A bias-exchange approach to protein folding. J. Phys. Chem. B 111, 4553–4559 (2007).

    Article  CAS  Google Scholar 

  47. Branduardi, D., Bussi, G. & Parrinello, M. Metadynamics with adaptive gaussians. J. Chem. Theory Comput. 8, 2247–2254 (2012).

    Article  CAS  Google Scholar 

  48. Hess, B., Kutzner, C., van der Spoel, D. & Lindahl, E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 4, 435–447 (2008).

    Article  CAS  Google Scholar 

  49. Bonomi, M. et al. PLUMED: a portable plugin for free-energy calculations with molecular dynamics. Comput. Phys. Commun. 180, 1961–1972 (2009).

    Article  CAS  Google Scholar 

  50. Branduardi, D., Gervasio, F.L. & Parrinello, M. From A to B in free energy space. J. Chem. Phys. 126, 054103 (2007).

    Article  Google Scholar 

  51. Marinelli, F., Pietrucci, F., Laio, A. & Piana, S. A kinetic model of Trp-Cage folding from multiple biased molecular dynamics simulations. PLoS Comput. Biol. 5, e1000452 (2009).

    Article  Google Scholar 

  52. Biarnes, X., Pietrucci, F., Marinelli, F. & Laio, A. METAGUI: A VMD interface for analyzing metadynamics and molecular dynamics simulations. Comput. Phys. Commun. 183, 203–211 (2012).

    Article  CAS  Google Scholar 

  53. Corbi-Verge, C. et al. Two-state dynamics of the SH3-SH2 tandem of Abl kinase and the allosteric role of the N-cap. Proc. Natl. Acad. Sci. USA 110, E3372–E3380 (2013).

    Article  CAS  Google Scholar 

  54. Daura, X. et al. Peptide folding: when simulation meets experiment. Angew. Chem. Int. Ed. 38, 236–240 (1999).

    Article  CAS  Google Scholar 

  55. Branduardi, D., Marinelli, F. & Faraldo-Gómez, J.D. Atomic-resolution dissection of the energetics and mechanism of isomerization of hydrated ATP-Mg2+ through the SOMA string method. J. Comput. Chem. 37, 575–586 (2016).

    Article  CAS  Google Scholar 

  56. Enright, A.J., Van Dongen, S. & Ouzounis, C.A. An efficient algorithm for large-scale detection of protein families. Nucleic Acids Res. 30, 1575–1584 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The experimental results reported in this article derive from measurements made at the Advanced Photon Source of the Argonne National Laboratory, GM/CA (23ID), which is operated by the University of Chicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. We thank the beamline staff for their assistance in data collection. This work was supported in part by the Howard Hughes Medical Institute; by grants from the National Institutes of Health (R01GM079179 to Y.J.) and the Welch Foundation (grant I-1578 to Y.J.); by the General Program of the National Natural Science Foundation of China (project 31470817 to J.L.); and by the Division of Intramural Research of the National Heart, Lung and Blood Institute, National Institutes of Health (to F.M. and J.D.F.-G.).

Author information

Authors and Affiliations

Authors

Contributions

J.L. and Y.J. designed the experimental studies and analyzed the resulting data. J.L., C.L. and Y.H. performed the experimental research. F.M. and J.D.F.-G. designed the computational research and analyzed the corresponding data. F.M. performed the computational work. J.L., Y.J., F.M. and J.D.F.-G. wrote the paper.

Corresponding authors

Correspondence to Jun Liao, José D Faraldo-Gómez or Youxing Jiang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Architecture of the Na+/Ca2+ exchanger and atomic structure of the outward-facing Na+-loaded state.

(a) Topology of the transmembrane functional unit of NCX. The so-called α-repeats are highlighted with solid colors (orange, blue). Eukaryotic NCX homologs contain a large intracellular regulatory domain between TM5 and TM6, whereas in microbial NCX homologs TM5 and TM6 are linked by a short loop. As shown in Figs. 2 and 4, in NCX_Mj the N-terminal half of TM7 is a straight helix (TM7ab) when the Sext binding site is empty, but is bent into two short helices (TM7a and 7b) when Na+ binds to Sext and this site becomes occluded. It is plausible that similar changes take place in the symmetry-related TM2, in the inward-facing states. (b) Crystal structure of NCX_Mj at high Na+ concentration (PDB code 3V5U) (Liao, J. et al. Structural insight into the ion-exchange mechanism of the sodium/calcium exchanger. Science 335, 686-690, 2012. Green and red spheres represent Na+ and water, respectively. The N- and C-terminal halves are colored yellow and cyan, respectively. (c) Highlight of the two α-repeats flanking the four central binding sites. TM2 and TM3 form α1 (yellow) and TM7 and TM8 form α2 (cyan). (d) Atomic details of the four binding sites, termed Sext, SCa, Smid and Sint. Residues that participate in Na+ chelation are indicated (with carbonyl oxygen atoms in red). Insets show the ion-coordination structure at each site. At Sext, SCa, and Sint, the ion-coordination numbers (5 or 6) and ion-ligand distances (~2.4 Å) are optimal for Na+ binding. The Smid site has a coordination number of 4 and ion-ligand distances of 2.6 Å and 2.9 Å, resulting in a calculated valence of far less than 1.0 for Na+; it is therefore not optimal for Na+, and thus the density at this site was assigned to a water molecule. Asp240 at Smid is suggested to be protonated, based on previous simulation and functional studies (Marinelli, F. et al. Proc. Natl. Acad. Sci. USA 111, E5354-62, 2014).

Supplementary Figure 2 Sr2+ and Ca2+ binding to NCX_Mj in a partially open outward-facing conformation.

(a) Titration of NCX_Mj crystals with decreasing amounts of Sr2+ at low Na+ concentration. The changes in Sr2+ occupancy are shown with Fo-Fc ion-omit maps calculated to 2.8 Å using data scaled to a common reference, and contoured at 7σ (solid-line panels). The map in the inset (dashed line) was calculated to 2.55 Å and contoured at 4σ, to reveal the bound Na+ (green spheres) and water (red sphere) observed at low Sr2+ concentration. (b) Titration with increasing amounts of Na+ at high Sr2+ concentration. The maps in the insets (dashed lines) were recalculated to 2.3 Å for a crystal soaked 10 mM Sr2+ and 10 mM Na+ and to 2.5 Å for a crystal soaked in 10 mM Sr2+ and 100 mM Na+, and contoured at 4σ, to reveal the bound Na+ (green spheres) and water (red sphere). At 10 mM Na+, the density reflects a mixture of partially bound Sr2+ at SCa, partially bound Na+ at Sint and at SCa, and a water molecule bound at Smid. At 100 mM Na+, the NCX_Mj structure reverts to the occluded state. (c) Titrations with Ca2+. At 10 mM, Ca2+ binds either to SCa or Smid, with SCa slightly preferred. At 0.1 mM Ca2+, the density distribution in the ion binding sites is virtually the same as that in the 2.5 mM Na+ only condition (Fig. 1c) indicating no bound Ca2+. Increasing Na+ to 10 mM similarly diminishes Ca2+ occupancy. All Fo-Fc ion omit maps (except those in the inset) were calculated to 2.65 Å using data scaled to a common reference and contoured at 5σ. The map in the inset is calculated at 2.2 Å and contoured at 4σ, and demonstrates that the density detected in the crystal soaked in 10 mM Ca2+ and 10 mM Na+ is a mixture of partially bound Ca2+ and Na+.

Supplementary Figure 3 Statistical survey of Ca2+-binding sites in the Protein Data Bank featuring two concurrent ion-carboxyl interactions.

(a) Distributions of observed Ca2+ coordination numbers in binding sites where the carboxyl groups are arranged so as to either two, one or no bi-dentate interactions. The number of Ca2+-binding sites identified in each case are indicated. Note that for the double bi-dentate mode observed in NCX_Mj (E54 and E213), the most probable coordination number, N, is 8 (>90%), but N = 7 is also observed. (b) Orientational preference in observed Ca2+-binding sites with two opposing carboxylate interactions in a bi-dentate mode, for N = 7 or N = 8. The figure shows the most probable angle (Φ) formed by the two opposing carboxyl groups (average ± STD in orange and grey, respectively), as well as the minimum distance between them, on average. The near orthogonal arrangement of E54 and E213 in NCX_Mj (green), and the separation between these groups, are characteristic of binding sites with N = 8. This analysis includes X-ray structures deposited in the PDB with less than 70% of sequence identity and with resolution higher than 2.5 Å. The analysis includes only Ca2+ binding sites that appear to be complete; a binding site is considered to be incomplete if the geometric center of the coordinating atoms deviates from the Ca2+ ion by more than 0.4 Å. (Note this threshold value is two times the standard deviation of this distance for binding sites in which the coordination number is 7 or 8). The coordinating atoms are defined as those within 3.0 Å of the ion, excluding hydrogen or carbon atoms, and other ions.

Supplementary Figure 4 Predicted structures of the Na+- and Ca2+-bound states of outward-facing, occluded NCX_Mj, contrasted with mutagenesis data.

(a-b) Close-up view of the predicted configuration of the ion-binding region of NCX_Mj, in the transport-ready Na+- and Ca2+-bound states. Note the striking symmetry of this region, relative to the membrane mid-plane, despite the fact the transporter adopts an outward-facing (i.e. asymmetric) conformation. The predictions derive from MD simulations and from a statistical survey of Ca2+-binding sites in the Protein Data Bank. Only side-chains flanking the four binding sites are highlighted. The dominant hydrogen-bonding patterns observed in the simulations are also indicated. A water-density iso-surface calculated from the simulation data is shown as a grey mesh, to indicate the degree of occlusion of the binding sites from the extracellular solution. (c-d) Residues lining the ion-binding sites in NCX_Mj whose mutation inactivates or significantly inhibits the activity of NCX_Mj or NCX1.1. (75% of the binding-site residues are identical in these two proteins, while the remaining 25% are homologous). Labels “A” and “B” indicate residues in NCX_Mj whose mutation to alanine inactivates the Na+-Ca2+ or Ca2+-Ca2+ exchange mechanism, respectively (<25% of wild-type activity), according to 45Ca2+ uptake assays of overexpressed NCX_Mj in right-side-out E. coli membrane vesicles (Marinelli, F. et al. Proc. Natl. Acad. Sci. USA 111, E5354-62, 2014; Giladi, M. et al. Sci. Rep. 6, 20753, 2016). Labels “C” and “D” indicate residues in NCX_Mj for which alanine mutation reduces the Na+-Ca2+ or Ca2+-Ca2+ exchange activity respectively (<50% of wild-type), according to the same measurements. The label “a” indicates residues in NCX1.1 whose mutation to cysteine inactivates or significantly inhibits the transport mechanism, as reported by patch-clamp recordings of the currents elicited upon Na+/Ca2+ exchange, either in the direct (Na+ inwards, Ca2+ outwards) or reverse mode (John, S. A. et al. Proc. Natl. Acad. Sci. USA 110, 7500-7505, 2013). The label “b” reflects comparable effects according to 45Ca2+ uptake experiments of NCX1.1, upon mutation of Ser to Ala or Cys, Thr to Ala, Glu to Gln, Asp to Asn and Asn to Val (Nicoll, D. A. et al. J Biol Chem 271, 13385-13391, 1996). The label “c” also reflects 45Ca2+ uptake measurements, for cysteine mutations (Iwamoto, T. et al. J. Biol. Chem. 275, 38571-38580, 2000). Lastly, label “d” refers to the inactivating effect of the S139A and N143V mutations in NCX1.1 (homologous to S77 and N81 of NCX_Mj), according to activity assays based on patch-clamp recordings and 45Ca2+ uptake measurements (Ottolia, M. et al. J. Biol. Chem. 280, 1061-1069, 2005). These experiments also showed that N143D and N842D (N81 and D240 in NCX_Mj) are inhibited but regain significant Na+/Ca2+ exchange activity, consistent with our proposal that Smid is not a functional ion-binding site, and that protonation of aspartate residues flanking this site is required for transport (Marinelli, F. et al. Proc. Natl. Acad. Sci. USA 111, E5354-62, 2014; Giladi, M. et al. Sci. Rep. 6, 20753, 2016).

Supplementary Figure 5 Assessment and correction of the CHARMM27 parameters representing the interactions of Ca2+ with water and with carboxyl groups.

(a) Structure of hydrated Ca2+, in terms of the Ca2+-water-oxygen radial distribution function (solid line, black) and the cumulative coordination number (dashed line, red). The data derives from a 20-ns MD simulation at 298 K and 1 bar, using the CHARMM27 force-field and 1,470 water molecules. Experimental ranges from neutron diffraction and EXAFS experiments (Hewish, N.A. et al. Nature 297, 138-139, 1982; Jalilehvand, F. et al. J. Am. Chem. Soc. 123, 431-441, 2001; Fulton, J. L., et al. J. Phys. Chem. A 107, 4688-4696, 2003) are indicated (grey bands) for comparison. (b) Optimization of the CHARMM27 force-field parameters describing the non-bonded interaction between Ca2+ and acetate. The plot shows calculated potential-of-mean-force (PMF) profiles as a function of the distance r between Ca2+ and the central carbon atom in acetate. The corresponding dissociation constants Kd are indicated, along with the experimental values (Daniele, P.G. et al. Coordin. Chem. Revs. 252, 1093-1107, 2008). The PMF was set to zero at a distance of 12 Å. Alternative PMF profiles were computed for different values of the Lennard–Jones Rmin parameter specifically used for the interaction between Ca2+ and the acetate carboxyl–oxygen atoms. The default value in CHARMM27 is 3.067 Å, which results in Kd values that are 3-4 orders of magnitude smaller than the experimental values; the Rmin value used in this study to simulate the Ca2+-bound NCX_Mj is 3.23 Å. To derive the Kd values, the PMF profiles are integrated over the range in r that encompasses both the contact ion-pair (CIP) and the solvent-shared ion-pair (SIP) complexes, i.e., up to Roff = 6.2 Å. (c) Ca2+-water cumulative coordination number in the context of the bi-dentate acetate-Ca2+ CIP, for different values of the Rmin parameter used for the Ca2+-carboxyl–oxygen interaction; the number of water molecules in the first hydration shell is 5, irrespective of the value of Rmin.

Supplementary Figure 6 Assessment of the relative magnitude of ion-charge transfer effects in NCX_Mj and in solution.

Molecular systems considered for (a) Ca2+, (b) Na+ and (c) Sr2+. The value reported, ΔΔq, is the amount of charge (in units of e) that is transferred from each of the cations to the protein, in excess to what is observed for the water clusters (which feature the most probably hydration structure in solution). The results are based on quantum-mechanical calculations of the electronic structure of each system. For Na+, the value indicated in the figure is for the SCa site in the 3 × Na+, occluded state of NCX_Mj. The ΔΔq value for the SCa site in the 2 × Na+ semi-open state is similarly small. The ΔΔq values for the Sext and Sint sites are however ~0.1e.

Supplementary Figure 7 Estimated standard errors in the conformational free-energy landscapes of outward-facing NCX_Mj in different ion-occupancy states.

The two-dimensional maps shown correspond to those in Figs. 5, 6. For each simulation system, the error map was estimated by comparing two free-energy maps calculated from two halves of the simulation data. As shown, the typical statistical error is smaller than 1 kcal/mol, and slightly larger errors are found only in the high-free energy regions.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1 and 2, and Supplementary Notes 1–4 (PDF 3267 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liao, J., Marinelli, F., Lee, C. et al. Mechanism of extracellular ion exchange and binding-site occlusion in a sodium/calcium exchanger. Nat Struct Mol Biol 23, 590–599 (2016). https://doi.org/10.1038/nsmb.3230

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3230

This article is cited by

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