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The structure of a calsequestrin filament reveals mechanisms of familial arrhythmia

Nature Structural & Molecular Biology volume 27, pages 1142–1151 (2020)Cite this article

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Abstract

Mutations in the calcium-binding protein calsequestrin cause the highly lethal familial arrhythmia catecholaminergic polymorphic ventricular tachycardia (CPVT). In vivo, calsequestrin multimerizes into filaments, but there is not yet an atomic-resolution structure of a calsequestrin filament. We report a crystal structure of a human cardiac calsequestrin filament with supporting mutational analysis and in vitro filamentation assays. We identify and characterize a new disease-associated calsequestrin mutation, S173I, that is located at the filament-forming interface, and further show that a previously reported dominant disease mutation, K180R, maps to the same surface. Both mutations disrupt filamentation, suggesting that disease pathology is due to defects in multimer formation. An ytterbium-derivatized structure pinpoints multiple credible calcium sites at filament-forming interfaces, explaining the atomic basis of calsequestrin filamentation in the presence of calcium. Our study thus provides a unifying molecular mechanism through which dominant-acting calsequestrin mutations provoke lethal arrhythmias.

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Fig. 1: Autosomal dominant CASQ2 disease mutations disrupt calsequestrin multimerization.
Fig. 2: Helical domain architecture of the cardiac calsequestrin filament.
Fig. 3: Cation binding leads to conformational shifts in calsequestrin dimers.
Fig. 4: Cations are trapped at inter-dimer filament-forming interfaces.
Fig. 5: The cardiac calsequestrin filament contains a continuous, solvent-accessible lumen along its long axis.
Fig. 6: Dominant disease-associated mutations disrupt the CASQ2 filament-forming interface.
Fig. 7: Heterozygous mutations at different calsequestrin interfaces display different inheritance patterns of disease.

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Data availability

The structures determined as part of this work are deposited in the Protein Data Bank (PDB) under identifiers 6OWV (native) and 6OWW (ytterbium-soaked). The raw diffraction dataset for the native structure is deposited in Zenodo under https://doi.org/10.5281/zenodo.2941360. The raw diffraction dataset for the structure in complex ith ytterbium is likewise deposited in Zenodo under https://doi.org/10.5281/zenodo.2943248. All data presented in the manuscript are also available at https://github.com/errontitus/casq2-structure-function. Source data are provided with this paper.

Code availability

Code to generate all results and figures from the manuscript is available at https://github.com/errontitus/casq2-structure-function. The manuscript and all figure layouts were constructed entirely in LaTeX using PGF/TikZ.

References

  1. Bers, D. M. Macromolecular complexes regulating cardiac ryanodine receptor function. J. Mol. Cell. Cardiol. 37, 417–429 (2004).

    CAS  PubMed  Google Scholar 

  2. Royer, L. & Ríos, E. Deconstructing calsequestrin. Complex buffering in the calcium store of skeletal muscle. J. Physiol. 587, 3101–3111 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Guerrero-Hernández, A. et al. in Calcium Signaling (ed Islam, M. S.) 337–370 (Springer International Publishing, 2020).

  4. MacLennan, D. H. & Wong, P. T. Isolation of a calcium-sequestering protein from sarcoplasmic reticulum. Proc. Natl Acad. Sci. USA 68, 1231–1235 (1971).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. MacLennan, D. H. Isolation of a second form of calsequestrin. J. Biol. Chem. 249, 980–984 (1974).

    CAS  PubMed  Google Scholar 

  6. Ostwald, T. J. & MacLennan, D. H. Isolation of a high affinity calcium-binding protein from sarcoplasmic reticulum. J. Biol. Chem. 249, 974–979 (1974).

    CAS  PubMed  Google Scholar 

  7. Costello, B. et al. Characterization of the junctional face membrane from terminal cisternae of sarcoplasmic reticulum. J. Cell Biol. 103, 741–753 (1986).

    CAS  PubMed  Google Scholar 

  8. Franzini-Armstrong, C., Kenney, L. J. & Varriano-Marston, E. The structure of calsequestrin in triads of vertebrate skeletal muscle: a deep-etch study. J. Cell Biol. 105, 49–56 (1987).

    CAS  PubMed  Google Scholar 

  9. Wang, S. et al. Crystal structure of calsequestrin from rabbit skeletal muscle sarcoplasmic reticulum. Nat. Struct. Biol. 5, 476–483 (1998).

    CAS  PubMed  Google Scholar 

  10. Park, H. et al. Comparing skeletal and cardiac calsequestrin structures and their calcium binding: a proposed mechanism for coupled calcium binding and protein polymerization. J. Biol. Chem. 279, 18026–18033 (2004).

    CAS  PubMed  Google Scholar 

  11. Knollmann, B. C. et al. Casq2 deletion causes sarcoplasmic reticulum volume increase, premature Ca2+ release, and catecholaminergic polymorphic ventricular tachycardia. J. Clin. Invest. 116, 2510–2520 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Perni, S., Close, M. & Franzini-Armstrong, C. Novel details of calsequestrin gel conformation in situ. J. Biol. Chem. 288, 31358–31362 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Milstein, M. L., Houle, T. D. & Cala, S. E. Calsequestrin isoforms localize to different ER sub-compartments: evidence for polymer and heteropolymer-dependent localization. Exp. Cell Res. 315, 523–534 (2009).

    CAS  PubMed  Google Scholar 

  14. McFarland, T. P., Milstein, M. L. & Cala, S. E. Rough endoplasmic reticulum to junctional sarcoplasmic reticulum trafficking of calsequestrin in adult cardiomyocytes. J. Mol. Cell. Cardiol. 49, 556–564 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Knollmann, B. C. A “rough” journey to the sarcoplasmic reticulum—implications of altered calsequestrin trafficking for cardiac arrhythmia. J. Mol. Cell. Cardiol. 49, 554–555 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Sanchez, E. J., Lewis, K. M., Munske, G. R., Nissen, M. S. & Kang, C. Glycosylation of skeletal calsequestrin: implications for its function. J. Biol. Chem. 287, 3042–3050 (2012).

    CAS  PubMed  Google Scholar 

  17. Kirchhefer, U. et al. The human CASQ2 mutation K206N is associated with hyperglycosylation and altered cellular calcium handling. J. Mol. Cell. Cardiol. 49, 95–105 (2010).

    CAS  PubMed  Google Scholar 

  18. Zhang, L., Kelley, J., Schmeisser, G., Kobayashi, Y. M. & Jones, L. R. Complex formation between junctin, triadin, calsequestrin, and the ryanodine receptor. Proteins of the cardiac junctional sarcoplasmic reticulum membrane. J. Biol. Chem. 272, 23389–23397 (1997).

    CAS  PubMed  Google Scholar 

  19. Rani, S., Park, C. S., Sreenivasaiah, P. K. & Kim, D. H. Characterization of Ca2+-dependent protein-protein interactions within the Ca2+ release units of cardiac sarcoplasmic reticulum. Mol. Cells 39, 149–155 (2016).

    CAS  PubMed  Google Scholar 

  20. Handhle, A. et al. Calsequestrin interacts directly with the cardiac ryanodine receptor luminal domain. J. Cell Sci. 129, 3983–3988 (2016).

  21. Lewis, K. M., Ronish, L. A., Ríos, E. & Kang, C. Characterization of two human skeletal calsequestrin mutants implicated in malignant hyperthermia and vacuolar aggregate myopathy. J. Biol. Chem. 290, 28665–28674 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Bal, N. C. et al. The catecholaminergic polymorphic ventricular tachycardia mutation R33Q disrupts the N-terminal structural motif that regulates reversible calsequestrin polymerization. J. Biol. Chem. 285, 17188–17196 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Bal, N. C. et al. Probing cationic selectivity of cardiac calsequestrin and its CPVT mutants. Biochem. J. 435, 391–399 (2011).

    CAS  PubMed  Google Scholar 

  24. Gray, B. et al. A novel heterozygous mutation in cardiac calsequestrin causes autosomal dominant catecholaminergic polymorphic ventricular tachycardia. Heart Rhythm 13, 1652–1660 (2016).

    PubMed  PubMed Central  Google Scholar 

  25. Kim, E. et al. Characterization of human cardiac calsequestrin and its deleterious mutants. J. Mol. Biol. 373, 1047–1057 (2007).

    CAS  PubMed  Google Scholar 

  26. Sanchez, E. J., Lewis, K. M., Danna, B. R. & Kang, C. High-capacity Ca2+ binding of human skeletal calsequestrin. J. Biol. Chem. 287, 11592–11601 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Lewis, K. M. et al. Characterization of post-translational modifications to calsequestrins of cardiac and skeletal muscle. Int. J. Mol. Sci. 17, 1539 (2016).

  28. Krause, K. H., Milos, M., Luan-Rilliet, Y., Lew, D. P. & Cox, J. A. Thermodynamics of cation binding to rabbit skeletal muscle calsequestrin. Evidence for distinct Ca2+- and Mg2+-binding sites. J. Biol. Chem. 266, 9453–9459 (1991).

    CAS  PubMed  Google Scholar 

  29. Sawyer, L. & James, M. N. G. Carboxyl–carboxylate interactions in proteins. Nature 295, 79–80 (1982).

    CAS  PubMed  Google Scholar 

  30. Frederick, K. K., Marlow, M. S., Valentine, K. G. & Wand, A. J. Conformational entropy in molecular recognition by proteins. Nature 448, 325–329 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Milos, M., Schaer, J. J., Comte, M. & Cox, J. A. Calcium–proton and calcium-magnesium antagonisms in calmodulin: microcalorimetric and potentiometric analyses. Biochemistry 25, 6279–6287 (1986).

    CAS  PubMed  Google Scholar 

  32. Kamp, F., Donoso, P. & Hidalgo, C. Changes in luminal pH caused by calcium release in sarcoplasmic reticulum vesicles. Biophys. J. 74, 290–296 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Hidalgo, C., Donoso, P. & Rodriguez, P. H. Protons induce calsequestrin conformational changes. Biophys. J. 71, 2130–2137 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Winter, G. et al. DIALS: implementation and evaluation of a new integration package. Acta Crystallogr D Struct. Biol. 74, 85–97 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    CAS  Google Scholar 

  36. Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    PubMed  Google Scholar 

  38. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  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).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Terwilliger, T. SOLVE and RESOLVE: automated structure solution, density modification and model building. J. Synchrotron Radiat. 11, 49–52 (2004).

    CAS  PubMed  Google Scholar 

  43. Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64, 61–69 (2008).

    CAS  PubMed  Google Scholar 

  44. Zwart, P. H., Grosse-Kunstleve, R. W. & Adams, P. D. Xtriage and Fest: automatic assessment of X-ray data and substructure structure factor estimation. CCP4 Newsl. 43, 27–35 (2005).

    Google Scholar 

  45. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Lebedev, A. A. & Isupov, M. N. Space-group and origin ambiguity in macromolecular structures with pseudo-symmetry and its treatment with the program Zanuda. Acta Crystallogr. D Biol. Crystallogr. 70, 2430–2443 (2014).

    CAS  PubMed  Google Scholar 

  47. Dolinsky, T. J., Nielsen, J. E., McCammon, J. A. & Baker, N. A. PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 32, W665–W667 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Cornell, W. D. et al. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 117, 5179–5197 (1995).

    CAS  Google Scholar 

  49. Jurrus, E. et al. Improvements to the APBS biomolecular solvation software suite. Protein Sci. 27, 112–128 (2018).

    CAS  PubMed  Google Scholar 

  50. Schrödinger, LLC. The PyMOL Molecular Graphics System, Version 2.2.3 (2019).

  51. Ho, B. K. & Gruswitz, F. HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 8, 49 (2008).

    PubMed  PubMed Central  Google Scholar 

  52. Beitz, E. TeXshade: shading and labeling of multiple sequence alignments using LaTeX2e. Bioinformatics 16, 135–139 (2000).

    CAS  PubMed  Google Scholar 

  53. Hunter, J. D. Matplotlib: a 2D graphics environment. Comput. Sci. Eng. 9, 90–95 (2007).

    Google Scholar 

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Acknowledgements

We thank the patient and the patient’s family. We thank J. Roberts for expert clinical review. We thank C. Agnew, J. Fraser, J. Holton, L. Liu, M. Lolicato, B. Mensa and T. Thaker for technical assistance with crystallographic data collection and processing. We thank L. Liu and T. Thaker for technical assistance with protein preparation and crystallization screens. We thank M. Grabe, A. Kao, and O. Rosenberg for helpful discussions. This work was supported by NIH/NHLBI grant F30 HL137329 (E.W.T.), a Sarnoff Foundation Fellowship (E.W.T), NIH/NIGMS grant T32 GM007618 to the UCSF Medical Scientist Training Program (E.W.T), NIH/NHLBI grant DP2 HL123228 (R.C.D.) and American Heart Association grant 17IRG33460152 (R.C.D.). Clinical data collection and genetic testing were supported by the UCSF Comprehensive Genetic Arrhythmia Program. Beamline 8.3.1 at the Advanced Light Source is operated by the University of California Office of the President, Multicampus Research Programs and Initiatives grant MR-15-328599, the National Institutes of Health (R01 GM124149 and P30 GM124169), Plexxikon Inc., and the Integrated Diffraction Analysis Technologies program of the US Department of Energy Office of Biological and Environmental Research. The Advanced Light Source (Berkeley, CA) is a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the US Department of Energy under contract number DEAC02-05CH11231, Office of Basic Energy Sciences.

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Author notes

  1. Rahul C. Deo

    Present address: Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Boston, MA, USA

Authors and Affiliations

  1. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA, USA

    Erron W. Titus, Frederick H. Deiter, Chenxu Shi, Natalia Jura & Rahul C. Deo

  2. Department of Medicine, University of California, San Francisco, San Francisco, CA, USA

    Julianne Wojciak, Melvin Scheinman & Rahul C. Deo

  3. Cardiovascular Genetics Program, University of California, San Francisco, San Francisco, CA, USA

    Julianne Wojciak, Melvin Scheinman & Rahul C. Deo

  4. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA

    Natalia Jura

  5. Harvard Medical School, Harvard University, Boston, MA, USA

    Rahul C. Deo

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Contributions

R.C.D. and E.W.T conceived and designed the study. R.C.D., N.J. and E.W.T. designed and oversaw experiments. E.W.T., F.H.D. and C.S. performed experiments. M.S. and J.W. collected and analyzed clinical data. E.W.T. analyzed experimental data. E.W.T. wrote the manuscript. M.S., J.W., N.J. and R.C.D. reviewed and edited the manuscript.

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Correspondence to Erron W. Titus or Rahul C. Deo.

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The authors declare no competing interests.

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Peer review information Beth Moorefield and Katarzyna Marcinkiewicz were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Turbidity assay controls; related to Fig. 1.

a, Stoichiometric addition of EDTA demonstrates immediate reversal of calcium-induced turbidity. b, Turbidity assay for the S173I mutant in 0 mM KCl. Error bars represent the mean ± s.d of n=3 technical replicates. Data for graphs shown in panels (a, b) are available as source data.

Source data

Extended Data Fig. 2 The inter-dimer interface of the new candidate cardiac calsequestrin filament exhibits all-by-all contacts and greater buried surface area compared to all other published calsequestrin structures; related to Fig. 2.

For each published calsequestrin structure, the inter-dimer interface with the greatest buried surface area is shown. Residues with buried surface area at the interface are rendered as spheres. Where similar PDB codes are listed, inter-dimer interfaces are roughly isomorphous with the example structure shown, although the space group and unit cell used to determine the structure sometimes differ.

Extended Data Fig. 3 The 3-Helix configuration of the new calsequestrin filament candidate promotes close packing of thioredoxin domains.

a, The candidate cardiac calsequestrin filament assembled from crystallographic symmetry operations on PDB ID 6OWV (human CASQ2, this study). The filament exhibits tight packing of protomers and thioredoxin domains (shown on the right using equal-size spheres placed at the center of mass of each thioredoxin domain). b, A putative skeletal calsequestrin filament assembled from crystallographic symmetry operations on PDB ID 1A8Y (rabbit CASQ1, 1998). Right-side: equal-size spheres represent thioredoxin domains. c, A putative skeletal calsequestrin filament assembled from crystallographic symmetry operations on PDB ID 1SJI (canine CASQ2, 2005). Right-side: equal-size spheres represent thioredoxin domains.

Extended Data Fig. 4 Electron density and anomalous difference maps for Yb-binding sites at the cardiac calsequestrin intra-dimer interface; related to Fig. 3.

a, Electron density (blue mesh) and anomalous difference maps for the D140-E143-E147 region. b, Electron density and anomalous difference maps for the D310 region.

Extended Data Fig. 5 Dimer overlays reveal that calsequestrin structures can be classified into tightly packed or loosely packed dimers; related to Fig. 3.

Dimers from published calsequestrin structures (lighter orange and green) are overlaid onto the tightly packed dimer from the present study (6OVW, darker orange and green). In each dimer pair, chain A is aligned to chain A to illustrate the relative displacement of chain B. The concentration of divalent cations used in the crystallization conditions is noted below. The overlays reveal two distinct conformational groupings. The more tightly packed conformation with inwardly rotated chains resembles the dimer in this study and appears to form at low pH or in the presence of neutralizing divalents.

Extended Data Fig. 6 Tightly packed calsequestrin dimers consistently exhibit increased conformational disorder in domain I; related to Fig. 3.

The top panel shows tightly packed calsequestrin dimers (that is dimers of calsequestrin crystallized in low pH or with high concentration of multivalent cations). In these structures, solvent-exposed loops in domain I are consistently disordered. In PDB 6OVW, the disordered loop region is omitted due to the high level of disorder. This same region (boxed, residues 58-68) is highly disordered in similar structures. The bottom panel shows loosely packed calsequestrin dimers (that is dimers of calsequestrin crystallized at neutral pH with low or trace concentrations of multivalent cations). The resolution for each structure is indicated, and several structures of non-comparable resolution are excluded (2VAF, 5CRE, 5KN0).

Extended Data Fig. 7 Electron density and anomalous difference maps for Yb-binding Sites at the cardiac calsequestrin filament’s inter-dimer interface; related to Fig. 4.

a, Electron density and anomalous difference maps for the D144-E174 region of interest. b, Electron density and anomalous difference maps for the D50-K180-E184-E187 region. c, Electron density and anomalous difference maps for the D348-D350 region. d, Electron density and anomalous difference maps for the D351-E357 region.

Extended Data Fig. 8 Turbidity assays showing effect of alanine mutagenesis of additional Yb-binding sites at the cardiac calsequestrin inter-dimer interface; related to Fig. 4.

a, Turbidity assay after alanine mutagenesis of the putative calcium-coordinating residues D348 and D350. b, Turbidity assay after alanine mutagenesis of the putative calcium-coordinating residues D351 and E357. Error bars represent the mean ± s.d of n=3 technical replicates. Data for graphs shown in panels (a, b) are available as source data.

Source data

Extended Data Fig. 9 Electron density map for a hydrophilic pocket at the cardiac calsequestrin filament’s inter-dimer interface; related to Fig. 6.

The S173 inter-dimer region of the calsequestrin filament with electron density shown as a blue mesh.

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Titus, E.W., Deiter, F.H., Shi, C. et al. The structure of a calsequestrin filament reveals mechanisms of familial arrhythmia. Nat Struct Mol Biol 27, 1142–1151 (2020). https://doi.org/10.1038/s41594-020-0510-9

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