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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.

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.

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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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