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Architecture and conformational switch mechanism of the ryanodine receptor

Nature volume 517, pages 3943 (01 January 2015) | Download Citation

Abstract

Muscle contraction is initiated by the release of calcium (Ca2+) from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors (RyRs). RyRs are homotetrameric channels with a molecular mass of more than 2.2 megadaltons that are regulated by several factors, including ions, small molecules and proteins. Numerous mutations in RyRs have been associated with human diseases. The molecular mechanism underlying the complex regulation of RyRs is poorly understood. Using electron cryomicroscopy, here we determine the architecture of rabbit RyR1 at a resolution of 6.1 Å. We show that the cytoplasmic moiety of RyR1 contains two large α-solenoid domains and several smaller domains, with folds suggestive of participation in protein–protein interactions. The transmembrane domain represents a chimaera of voltage-gated sodium and pH-activated ion channels. We identify the calcium-binding EF-hand domain and show that it functions as a conformational switch allosterically gating the channel.

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Primary accessions

Electron Microscopy Data Bank

Data deposits

The coordinates and electron microscopy density maps for closed and open states have been deposited in the RCSB Protein Data Bank under the accession codes 4UWA and 4UWE, and in the Electron Microscopy Data Bank under the accession codes EMD-2751 and EMD-2752, respectively.

References

  1. 1.

    in Calcium Signaling (ed. ) Ch. 1 1–25 (Springer, 2012)

  2. 2.

    & Sarcoplasmic reticulum calcium release compared in slow-twitch and fast-twitch fibres of mouse muscle. J. Physiol. (Lond.) 551, 125–138 (2003)

  3. 3.

    , , & Ryanodine receptors: structure, expression, molecular details, and function in calcium release. Cold Spring Harb. Perspect. Biol. 2, a003996 (2010)

  4. 4.

    & The structural biology of ryanodine receptors. Sci. China. Life Sci. 54, 712–724 (2011)

  5. 5.

    & Ryanodine receptor structure: progress and challenges. J. Biol. Chem. 284, 4047–4051 (2009)

  6. 6.

    Ryanodine receptors: allosteric ion channel giants. J. Mol. Biol. (15 August 2014)

  7. 7.

    & Inositol 1,4,5-trisphosphate and its receptors. Adv. Exp. Med. Biol. 740, 255–279 (2012)

  8. 8.

    Ryanodine receptor calcium channels and their partners as drug targets. Biochem. Pharmacol. 79, 1535–1543 (2010)

  9. 9.

    & Ryanodine receptor channelopathies. Pflugers Arch. 460, 467–480 (2010)

  10. 10.

    & Potassium channel structures: do they conform? Curr. Opin. Struct. Biol. 14, 440–446 (2004)

  11. 11.

    , , & The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule. Nature 468, 585–588 (2010)

  12. 12.

    et al. Structural determination of the phosphorylation domain of the ryanodine receptor. FEBS J. 279, 3952–3964 (2012)

  13. 13.

    , & Disease mutations in the ryanodine receptor central region: crystal structures of a phosphorylation hot spot domain. Structure 20, 1201–1211 (2012)

  14. 14.

    , , , & Topology of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (RyR1). Proc. Natl Acad. Sci. USA 99, 16725–16730 (2002)

  15. 15.

    , , & A census of protein repeats. J. Mol. Biol. 293, 151–160 (1999)

  16. 16.

    , , , & Calmodulin-binding locations on the skeletal and cardiac ryanodine receptors. J. Biol. Chem. 287, 30328–30335 (2012)

  17. 17.

    , , & Complex of calmodulin with a ryanodine receptor target reveals a novel, flexible binding mode. Structure 14, 1547–1556 (2006)

  18. 18.

    , & SPRY domains in ryanodine receptors (Ca2+-release channels). Trends Biochem. Sci. 22, 193–194 (1997)

  19. 19.

    , , , & Location of divergent region 2 on the three-dimensional structure of cardiac muscle ryanodine receptor/calcium release channel. J. Mol. Biol. 338, 533–545 (2004)

  20. 20.

    et al. Exploring the diversity of SPRY/B30.2-mediated interactions. Trends Biochem. Sci. 38, 38–46 (2013)

  21. 21.

    & When protein folding is simplified to protein coiling: the continuum of solenoid protein structures. Trends Biochem. Sci. 25, 509–515 (2000)

  22. 22.

    et al. Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature 339, 439–445 (1989)

  23. 23.

    et al. Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 265, 2244–2256 (1990)

  24. 24.

    , & Subunit structure of junctional feet in triads of skeletal muscle: a freeze-drying, rotary-shadowing study. J. Cell Biol. 99, 1735–1742 (1984)

  25. 25.

    , & Physical coupling between ryanodine receptor-calcium release channels. J. Mol. Biol. 349, 538–546 (2005)

  26. 26.

    , , & Mutational analysis of putative calcium binding motifs within the skeletal ryanodine receptor isoform, RyR1. J. Biol. Chem. 279, 53028–53035 (2004)

  27. 27.

    & EF-hand calcium-binding proteins. Curr. Opin. Struct. Biol. 10, 637–643 (2000)

  28. 28.

    et al. Solution structure of calcium-free calmodulin. Nature Struct. Biol. 2, 768–776 (1995)

  29. 29.

    , , & Evidence for a role of C-terminal amino acid residues in skeletal muscle Ca2+ release channel (ryanodine receptor) function. FEBS Lett. 412, 223–226 (1997)

  30. 30.

    , , & The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 (2011)

  31. 31.

    et al. Structural basis for Ca2+ selectivity of a voltage-gated calcium channel. Nature 505, 56–61 (2014)

  32. 32.

    , , & Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504, 107–112 (2013)

  33. 33.

    et al. Mechanism of activation gating in the full-length KcsA K+ channel. Proc. Natl Acad. Sci. USA 108, 11896–11899 (2011)

  34. 34.

    et al. Flexible architecture of IP3R1 by Cryo-EM. Structure 19, 1192–1199 (2011)

  35. 35.

    et al. Purified ryanodine receptor from rabbit skeletal muscle is the calcium-release channel of sarcoplasmic reticulum. J. Gen. Physiol. 92, 1–26 (1988)

  36. 36.

    , & Structural characterization of the RyR1–FKBP12 interaction. J. Mol. Biol. 356, 917–927 (2006)

  37. 37.

    , , , & Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy. Nature 466, 329–333 (2010)

  38. 38.

    & Gating of the native and purified cardiac SR Ca2+-release channel with monovalent cations as permeant species. Biophys. J. 67, 1484–1494 (1994)

  39. 39.

    et al. FKBP12 binding modulates ryanodine receptor channel gating. J. Biol. Chem. 276, 16931–16935 (2001)

  40. 40.

    , & Single channel activity of the ryanodine receptor calcium release channel is modulated by FK-506. FEBS Lett. 352, 369–374 (1994)

  41. 41.

    , & A procedure for purification of the ryanodine receptor from skeletal muscle. Membr. Biochem. 8, 133–145 (1989)

  42. 42.

    et al. Chapter 11 - Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464, 211–231 (2009)

  43. 43.

    et al. Movies of ice-embedded particles enhance resolution in electron cryo-microscopy. Structure 20, 1823–1828 (2012)

  44. 44.

    & Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003)

  45. 45.

    & SIGNATURE: a single-particle selection system for molecular electron microscopy. J. Struct. Biol. 157, 168–173 (2007)

  46. 46.

    et al. SPARX, a new environment for Cryo-EM image processing. J. Struct. Biol. 157, 47–55 (2007)

  47. 47.

    & Prevention of overfitting in cryo-EM structure determination. Nature Methods 9, 853–854 (2012)

  48. 48.

    et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature Methods 10, 584–590 (2013)

  49. 49.

    RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

  50. 50.

    , & Identifying conformational states of macromolecules by eigen-analysis of resampled cryo-EM images. Structure 19, 1582–1590 (2011)

  51. 51.

    , & Quantifying the local resolution of cryo-EM density maps. Nature Methods 11, 63–65 (2014)

  52. 52.

    et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

  53. 53.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  54. 54.

    & Comparison of Segger and other methods for segmentation and rigid-body docking of molecular components in cryo-EM density maps. Biopolymers 97, 742–760 (2012)

  55. 55.

    et al. UCSF chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

  56. 56.

    , & Accurate prediction of solvent accessibility using neural networks-based regression. Proteins 56, 753–767 (2004)

  57. 57.

    et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

  58. 58.

    , & Modeling of loops in protein structures. Protein Sci. 9, 1753–1773 (2000)

  59. 59.

    , & Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 32, W526–W531 (2004)

  60. 60.

    & Protein structure prediction on the Web: a case study using the Phyre server. Nature Protocols 4, 363–371 (2009)

  61. 61.

    , , & IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 21, 3433–3434 (2005)

  62. 62.

    & The HMMTOP transmembrane topology prediction server. Bioinformatics 17, 849–850 (2001)

  63. 63.

    , , & Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001)

  64. 64.

    , & PoreWalker: a novel tool for the identification and characterization of channels in transmembrane proteins from their three-dimensional structure. PLOS Comput. Biol. 5, e1000440 (2009)

  65. 65.

    The PyMOL Molecular Graphics System version 1.3r1 (Schrödinger, LLC, 2010)

  66. 66.

    et al. Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography. Mol. Cell. Proteomics 11, M111.014126 (2012)

  67. 67.

    , & Lysine-specific chemical cross-linking of protein complexes and identification of cross-linking sites using LC–MS/MS and the xQuest/xProphet software pipeline. Nature Protocols 9, 120–137 (2014)

  68. 68.

    et al. Identification of cross-linked peptides from large sequence databases. Nature Methods 5, 315–318 (2008)

  69. 69.

    et al. False discovery rate estimation for cross-linked peptides identified by mass spectrometry. Nature Methods 9, 901–903 (2012)

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Acknowledgements

We are grateful to O. Hofnagel for assistance with electron microscopy facilities and R. S. Goody for continuous support and useful comments on the manuscript. R.G.E. thanks R. Chaves and K. Willegems for assistance with particle selection. We gratefully acknowledge R. Matadeen and S. de Carlo (FEI Company) for image acquisition at the National Center for Electron Nanoscopy in Leiden (NeCEN) which is co-financed by grants from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (project 175.010.2009.001) and by the European Union’s Regional Development Fund through ‘Kansen voor West’ (project 21Z.014). This work was funded by Humboldt Foundation (to R.G.E.), by the ‘Deutsche Forschungsgemeinschaft’ Grant RA 1781/1-1 (to S.R.), the Max Planck Society (to S.R. and R.G.E.), VIB and Vrij Universiteit Brussel (to R.G.E.).

Author information

Affiliations

  1. Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany

    • Rouslan G. Efremov
    •  & Stefan Raunser
  2. Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), 1050 Brussels, Belgium

    • Rouslan G. Efremov
  3. Structural Biology Brussels, Vrije Universiteit Brussel (VUB), 1050 Brussels, Belgium

    • Rouslan G. Efremov
  4. Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland

    • Alexander Leitner
    •  & Ruedi Aebersold
  5. Faculty of Science, University of Zurich, 8057 Zurich, Switzerland

    • Ruedi Aebersold

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Contributions

R.G.E. designed the project and performed research, S.R. and R.G.E. managed the project, analysed data and wrote the manuscript, A.L. and R.A. performed cross-linking mass spectrometry experiments in the laboratory of R.A.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Rouslan G. Efremov or Stefan Raunser.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Text, Supplementary Table 1 and additional references.

Videos

  1. 1.

    Conformational changes between open and closed states

    The video shows morphs of cryo-EM maps and structures of RyR1 between closed and open states of the channel. First an overview of conformational changes is shown for a dimer. Then the video zooms on conformational changes at the cytoplasmic surface of the membrane where the ion gate is formed by a bundle of four inner helices (the structure is coloured similar to Figure 1) and conformational changes around EF-hand (purple). To better visualize conformational changes around the EF-hand the density maps and structures of a fragment of a RyR1 protomer were aligned to the α-solenoid 1 region preceding the EF-hand (orange, left from the EF-hand), see also Figure 3.

  2. 2.

    Multiple conformations of RyR1 in closed and open states

    Morphs of cryo-EM density maps between two extreme conformations of RyR1 in the closed state and between two conformational transition modes identified in the open state. See also Extended Data Figure 9.

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DOI

https://doi.org/10.1038/nature13916

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