The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule

Journal name:
Nature
Volume:
468,
Pages:
585–588
Date published:
DOI:
doi:10.1038/nature09471
Received
Accepted
Published online

Many physiological events require transient increases in cytosolic Ca2+ concentrations. Ryanodine receptors (RyRs) are ion channels that govern the release of Ca2+ from the endoplasmic and sarcoplasmic reticulum1. Mutations in RyRs can lead to severe genetic conditions that affect both cardiac and skeletal muscle, but locating the mutated residues in the full-length channel structure has been difficult2, 3. Here we show the 2.5Å resolution crystal structure of a region spanning three domains of RyR type 1 (RyR1), encompassing amino acid residues 1–559. The domains interact with each other through a predominantly hydrophilic interface. Docking in RyR1 electron microscopy maps4, 5 unambiguously places the domains in the cytoplasmic portion of the channel, forming a 240-kDa cytoplasmic vestibule around the four-fold symmetry axis. We pinpoint the exact locations of more than 50 disease-associated mutations in full-length RyR1 and RyR2. The mutations can be classified into three groups: those that destabilize the interfaces between the three amino-terminal domains, disturb the folding of individual domains or affect one of six interfaces with other parts of the receptor. We propose a model whereby the opening of a RyR coincides with allosterically coupled motions within the N-terminal domains. This process can be affected by mutations that target various interfaces within and across subunits. The crystal structure provides a framework to understand the many disease-associated mutations in RyRs that have been studied using functional methods, and will be useful for developing new strategies to modulate RyR function in disease states.

At a glance

Figures

  1. Overall structure of the RyR1 A, B and C domains.
    Figure 1: Overall structure of the RyR1 A, B and C domains.

    a, Overall structure of rabbit RyR1 for amino acid residues 1–559. This portion of RyR1 consists of domain A (blue; 1–205), domain B (green; 206–394) and domain C (red; 395–532). The remaining 27 residues are not visible in the electron density. Flexible loops are shown as dashed lines. For reference, α-helices (α1–α6) and β-strands (1–24) are numbered. b, Different view of a. c, Typical omit difference density map in a randomly selected region, contoured at 3σ.

  2. Docking of RyR1 ABC in the 9.6[thinsp]A RyR1 cryo-EM map.
    Figure 2: Docking of RyR1 ABC in the 9.6Å RyR1 cryo-EM map.

    a, View from the cytoplasmic side towards the endoplasmic reticulum, depicting domain A (blue), domain B (green) and domain C (red). b, Close-up lateral view from the four-fold symmetry axis. For clarity, only two monomers are shown. c, Docking of domain A alone (blue) or domain BC alone (red) yields a near-perfect superposition on the docking position for ABC (grey). The probability of recapitulating the A–BC interface by chance is <10−10 (see also Supplementary Discussion).

  3. Disease-associated mutations in RyR1 and RyR2.
    Figure 3: Disease-associated mutations in RyR1 and RyR2.

    a, Position of disease-associated mutations in RyR1 (red) and additional such mutations in RyR2 (blue); regions not associated with disease (grey). For clarity, mutations in flexible loops are not shown. The view is similar to Fig. 1a. b, Salt bridges at the A–C interface. Domain A (blue), domain B (green) and domain C (red) are shown. Hydrogen bonds are represented by dashed lines. Underlined residues are targets for disease-associated mutations. c, Interface 1 across ABC subunits, according to docking in the 10.3Å RyR1 cryo-EM. Disease-associated mutation positions and E321 are labelled. Flexible loops are shown as coloured dashed lines. The R329 side chain is shown in its most common rotamer. R317 undergoes hydrogen bonding with the E321 main chain. The inset shows the relative view.

  4. The
    Figure 4: The

    ABC interface is labile. a, Surface of RyR1 domain A (blue) showing the C36 sulphydryl (yellow), and representation of domain B (green). Selected residues from domain B are depicted as a grey stick structure, with oxygen atoms (red) and nitrogen atoms (blue) highlighted. C36 is inaccessible in the presence of domain B (Supplementary Fig. 5). b, Model showing the impact of disease-associated mutations (asterisks) on the motion of domains A, B and C (ovals). Interfaces with other RyR domains are indicated by black combs. The exact nature and range of motion of these domains on opening of the channel are not known. Disease-associated mutations target multiple interfaces, thus facilitating motion of the domains. c, Superposition of the RyR1 ABC structure (coloured) with the InsP3R BC structure (grey) bound to InsP3 (stick representation; PDB ID: 1N4K), based on domain B of each structure. Treating the InsP3R BC domain as a rigid unit shows a different relative orientation of domains B and C. This conformational difference might be induced by InsP3 binding to InsP3R.

Accession codes

Primary accessions

Protein Data Bank

References

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Author information

Affiliations

  1. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

    • Ching-Chieh Tung,
    • Paolo A. Lobo,
    • Lynn Kimlicka &
    • Filip Van Petegem

Contributions

C.-C.T. expressed, purified and crystallized the protein, and collected diffraction data. P.A.L. cloned several initial constructs and assisted with the melting curve analysis. L.K. prepared the disease-associated mutation, purified the corresponding protein and measured the melting curves. F.V.P. designed and supervised the experiments, collected diffraction data, solved the structure, performed the docking experiments, and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Atomic coordinates and structure factors for the RyR1 ABC structure have been deposited with the Protein Data Bank (http://www.rcsb.org) under accession code 2XOA.

Author details

Supplementary information

PDF files

  1. Supplementary Information (3.4M)

    The file contains a Supplementary Discussion, Supplementary Tables 1-3 and Supplementary Figures 1-7 with legends.

Movies

  1. Supplementary Movie 1 (9.3M)

    This movie file shows a rotation of the docked RyR1ABC crystal structure inside the 9.6Å cryoEM map of RyR1 in the closed state.

Additional data