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

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Abstract

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 2.5 Å 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.

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Figure 1: Overall structure of the RyR1 A, B and C domains.
Figure 2: Docking of RyR1 ABC in the 9.6 Å RyR1 cryo-EM map.
Figure 3: Disease-associated mutations in RyR1 and RyR2.
Figure 4: The

Accession codes

Primary accessions

Protein Data Bank

Data deposits

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.

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Acknowledgements

We thank the staff at beamline 08ID-1 of the Canadian Light source, the Stanford Synchrotron Radiation Lightsource, K. Beam for the rabbit RyR1 clone, K. Lau for assistance with preparing the figures and the movie file, and C. Ahern, E. Moore and R. Pancaroglu for comments on the manuscript. F.V.P. is funded by the Canadian Institutes of Health Research (CIHR) and the Heart and Stroke Foundation of Canada, and is a CIHR new investigator and a Michael Smith Foundation for Health Research Scholar.

Author information

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.

Correspondence to Filip Van Petegem.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains a Supplementary Discussion, Supplementary Tables 1-3 and Supplementary Figures 1-7 with legends. (PDF 3564 kb)

Supplementary Movie 1

This movie file shows a rotation of the docked RyR1ABC crystal structure inside the 9.6Å cryoEM map of RyR1 in the closed state. (MOV 9601 kb)

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Tung, C., Lobo, P., Kimlicka, L. et al. The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule. Nature 468, 585–588 (2010) doi:10.1038/nature09471

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