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Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution

Nature volume 517, pages 50–55 (2015)Cite this article

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Abstract

The ryanodine receptors (RyRs) are high-conductance intracellular Ca2+ channels that play a pivotal role in the excitation–contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5,000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryomicroscopy. Three previously uncharacterized domains, named central, handle and helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative-charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity-filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities.

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Figure 1: Overall structure and domain organization of the rabbit RyR1.
Figure 2: Hierarchical organization of the tetrameric RyR1.
Figure 3: Structure of the channel domain of RyR1.
Figure 4: The intra- and inter-domain contacts of the RyR1 channel domain may contribute to channel gating.

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Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The atomic coordinates of the RyR1–FKBP12 complex have been deposited in the Protein Data Bank with the accession code 3J8H. The 3.8 Å electron microscopy map has been deposited in the Electron Microscopy Data Bank with accession code EMD-2807.

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Acknowledgements

This work was supported by funds from the Ministry of Science and Technology of China (2015CB910101, 2011CB910501 and 2014ZX09507003006), National Natural Science Foundation of China (projects 31321062, 31130002 and 31125009), a European Union Marie Curie Fellowship (to X.B.), and the UK Medical Research Council (MC_UP_A025_1013, to S.H.W.S.). The research of N. Yan was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute and an endowed professorship from Bayer Healthcare.

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Author notes

  1. Zhen Yan, Xiao-chen Bai and Chuangye Yan: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Bio-membrane and Membrane Biotechnology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, 100084, China

    Zhen Yan, Zhangqiang Li, Wei Peng & Nieng Yan

  2. Ministry of Education Key Laboratory of Protein Science, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, 100084, China

    Zhen Yan, Chuangye Yan, Jianping Wu, Tian Xie & Yigong Shi

  3. Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, 100084, China

    Zhen Yan, Chuangye Yan, Jianping Wu, Zhangqiang Li, Tian Xie, Wei Peng, Xueming Li, Yigong Shi & Nieng Yan

  4. MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK ,

    Xiao-chen Bai & Sjors H. W. Scheres

  5. Department of Biophysics, the Health Science Center & Center for Protein Science, Peking University, Beijing, 100191, China

    Chang-cheng Yin

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Contributions

Y.S. and N.Y. conceived the project. Z.Y., X.B., X.L., S.H.W.S., Y.S. and N.Y. designed experiments. Z.Y., X.B., C.Y., J.W., Z.L., T.X., W.P. and X.L. performed the experiments. All authors analysed the data and contributed to manuscript preparation. N.Y. and Y.S. wrote the manuscript.

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Correspondence to Sjors H. W. Scheres, Yigong Shi or Nieng Yan.

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Extended data figures and tables

Extended Data Figure 1 Purification and cryo-EM analysis of the rabbit RyR1 complex bound to the modulator FKBP12.

a, The final step of purification of the rabbit RyR1 bound to FKBP12. Shown here are a size-exclusion chromatogram of the RyR1–FKBP12 complex (left) and a SDS–PAGE gel of the peak fractions visualized by Coomassie blue staining (right). The shaded fractions in the left panel were pooled for cryo-EM analysis. kDa, kilodaltons; UV, ultraviolet. b, A representative electron micrograph of the rabbit RyR1–FKBP12 complex. Scale bar, 20 nm. c, Two-dimensional class averages of the electron micrographs. d, Gold-standard FSC curves for the density maps. The overall resolution is estimated at 3.8 Å. e, Tilt-pair validation of the correctness of the map. Particles of RyR1 were imaged twice at 0° and 20° tilt angles. The position of each dot represents the direction and amount of tilting for a particle pair in polar coordinates. Blue and red dots correspond to in-plane and out-of-plane tilt transformations, respectively. Most of the blue dots cluster at a tilt angle of approximately 20°, which validates the structure. f, Angular distribution for the final reconstruction. Each sphere represents one view and the size of the sphere is proportional to the number of particles for that view. The azimuth angle only spans 90° because of the four-fold symmetry axis that runs from top to bottom. g, FSC curves between the model and the cryo-EM map. Shown here are the FSC curves between the final refined atomic model and the reconstruction from all particles (black), between the model refined in the reconstruction from only half of the particles and the reconstruction from that same half (FSCwork, green), and between that same model and the reconstruction from the other half of the particles (FSCtest, red). h, The overall electron microscopy density map of the RyR1–FKBP12 is colour-coded to indicate a range of resolutions. Much of the central region of RyR1 is resolved at resolutions better than the overall 3.8 Å, while the periphery of the structure is of much lower resolution. i, Electron microscopy density map for the channel domain of the rabbit RyR1.Two perpendicular views are shown. The electron microscopy density maps were generated in Chimera67.

Extended Data Figure 2 An illustration of the model-building procedures for the RyR1–FKBP12 complex into the electron microscopy density map.

a, The step-by-step procedures for the generation of the overall structural model and domain assignment. Detailed descriptions can be found in Methods. b, Correlation between the previously assigned sub-regions21,22 and the corresponding domains in our 3.8 Å structure. c, The boundaries of the identified domains in the current structure of RyR1. HD, helical domain. The P2 domain is a phosphorylation hotspot in RyRs. P1 shares homology with P2. The numbers below the domains indicate their sequence boundaries. Those labelled red were identified on the basis of well-defined electron microscopy density maps. It is of particular note that the three SPRY domains and P1 domain appear to be intertwined. See Extended Data Fig. 6b for the following structural descriptions. Each SPRY domain consists mainly of a β-sandwich. In addition to the core β-sandwich (residues 639–826), SPRY1 also contains two pairs of anti-parallel β-strands (residues 1466–1491, brown, and 1615–1634, magenta), the primary sequences of which are connected to those of SPRY3. Similarly, SPRY2 contains a pair of anti-parallel β-strands (residues 827–845, silver) from SPRY1. The sequences of SPRY2 are also interrupted by those of the P1 domain. Consequently, the N and C termini of the SPRY1–3 region (residues 639 and 1634) are both contained within the SPRY1 structure.

Extended Data Figure 3 Electron microscopy density maps for the domains whose atomic structural models were generated de novo.

a, The electron microscopy density maps for the handle domain. Representative density map for one helix in the handle domain is shown on the right. b, The electron microscopy density maps for the central domain. Left to right: the density maps for the overall domain, the U-motif, and one representative helical repeat in the central domain, respectively. c–g, The electron microscopy density maps for the segments in the channel domain. Shown here are the density maps for the pore forming elements (c), the selectivity filter (d), the luminal hairpin loop (e), the CTD shown in stereo views (f), and VSL domain (g). The maps, shown as blue mesh, are contoured at 4σ and made in PyMol. Representative bulky residues which were used to aid sequence assignment are shown as sticks and labelled.

Extended Data Figure 4 Sequence alignment of RyR orthologues.

Secondary structural elements are indicated above the sequence alignment and domains are coloured the same as the structures shown in the main text figures. The numbering of the secondary elements refers to their sequential positions within the corresponding domains. Invariant amino acids are shaded light grey. The GenInfo Identifier (GI) codes for the sequences from top to bottom: rabbit RyR1 (156119408), human RyR1 (113204615), human RyR2 (308153558) and human RyR3 (325511382).

Extended Data Figure 5 Continued sequence alignment of RyR orthologues.

Owing to the enormous size of the proteins, the sequence alignment was divided into two figures with an overlap at the helix 7b from the helical domain. Notably, it was predicted that several EF-hand domains may exist between residues 4252 and 4545 (ref. 4). The lack of electron microscopy density for these domains may indicate their intrinsic flexibility in the absence of Ca2+. The structure and mechanism of these putative EF-hand domains await further investigation.

Extended Data Figure 6 Organization of the cytoplasmic domains of RyR1.

a, The NTD (yellow) participates in tetramerization. Compared to the previously reported crystal structure of the NTD28, five additional α-helices (residues 560–631, orange) were identified in the electron microscopy structure. b, The structure of the SPRY1–3 domains. See Extended Data Fig. 2c for the sequence assignment of the three intertwined domains. Note that the N terminus of SPRY1 is preceded by the NTD, and its C terminus (magenta) is followed by the handle domain (cyan). c, Structure of the handle domain. The disordered sequences are indicated by dashed lines. d, The helical domain comprises two discontinuous sequence fragments, HD1 and HD2, which are disrupted by the P2 domain. e, The helical repeats in subdomain C of the NTD resemble the armadillo repeats. Shown here is a superposition with a designed armadillo protein (PDB code 4DB8). f, Structural superposition of the handle domain with β-catenin (PDB code 3IFQ) suggests that five pairs of helices, 1–2, 4–5, 8–9, 10–11 and 14–15, in the handle domain exhibit structural homology to the armadillo repeats. g, The helical repeats in the central domain are armadillo-like repeats. Shown here is the superposition of the central domain with the armadillo repeats of the anaphase-promoting complex (PDB code 3NMW). h, The armadillo-like repeats in the NTD, handle and central domains are joined end-to-end to form a superhelical assembly. The appearance of the superhelical assembly resembles a question mark. i, Each central domain directly interacts with two adjacent NTDs. The convex side of the helical repeats is involved in binding to the NTDs. Two close-up views are shown to highlight key residues that may form hydrogen bonds at the interfaces. j, The NTDs, central, handle and helical domains form multiple interfaces. Shown here are two adjacent NTDs (NTD and NTD′), one central domain, one handle domain, and the N-terminal fragment of the helical domain. k, SPRY2 bridges the spatial gap between the handle domain and HD2 from the adjacent protomer. l, FKBP12 is bound in a cleft formed by the handle, NTD and SPRY1/3 domains. m, An extended hydrophobic loop from the handle domain reaches into the ligand-binding pocket of FKBP12. A close-up view is shown to illustrate the residues that may mediate the interactions.

Extended Data Figure 7 Alignment of the channel domain sequences of RyR orthologues.

Secondary structural elements are indicated above the sequence alignment. Invariant amino acids are shaded in grey. The residues that may constitute potential cation-binding sites are in red. The C2H2 zinc-finger motif is highlighted by a green background. The residues whose mutations or deletions have been identified in patients are indicated with coloured circles below the sequences. The colour code is annotated at the bottom. CCD, central core disease; CPVT1, catecholaminergic polymorphic ventricular tachycardia type 1; CRD, core/rod disease; MHS, malignant hyperthermia susceptibility.

Extended Data Figure 8 Structural comparison of the RyR1 channel domain with representative tetrameric cation channels of known structures.

a, Structural comparison of the pore-forming elements from different tetrameric cation channels of known structures. Two diagonal protomers are shown. In all structures, the S5 and S6 segments are coloured grey. In the structures of CavAb and KcsA, the bound ions are shown as spheres. PDB codes: 4MS2 for CavAb, 3J5Q for TRPV1 and 1K4C for KcsA. b, Structural comparison of the transmembrane region of RyR1 VSL to the VSDs or like domains in the indicated tetrameric ion channels. Note that a hydrophilic sequence between S1 and S2 (residues 4579–4639) exhibits poor electron microscopy density and constitutes the least conserved DR1 region (DR for ‘divergent’) in the RyR1 channel domain11 (Extended Data Fig. 7). The ordered segments within this sequence form a pair of short anti-parallel β-strands that extends into the sarcoplasmic reticulum lumen. PDB codes: 2R9R for the Kv1.2/Kv2.1 paddle chimaera, 4DXW for NavRh, and 3J5P for TRPV1.

Extended Data Figure 9 Mapping of the disease-associated point mutations onto the structure of the RyR1 channel domain.

a, The residues that are targeted for disease-derived mutations are highlighted by different colours: purple blue for CCD, red for MHS, green for SM (samaritan myopathy), cyan for MMDO (minicore myopathy with ophthalmoplegia), yellow for CRD, dark purple for CFTD (congenital fibre type disproportion), and magenta for CPVT1. See Supplementary Table 1 for details of these mutations. b, Disease-related mutations in the handle domain. The concerned residues, which are positioned on the surface of the handle domain, may be involved in the interaction with modulators or other domains within RyR1. c, Disease-related residues aligning the inter-domain interface between the NTD, handle and central domains. d, Representative disease-related residues involved in the interaction between the central domain and channel domain. e, The channel domain represents a hotspot for mutations associated with several diseases. See Extended Data Fig. 7 and Supplementary Table 1 for details of the mutations.

Extended Data Figure 10 Intra- and inter-domain interactions that may be important for the long-range allosteric regulation of channel gating.

a, Extensive van der Waals interactions exist between the pore-forming segments, and between the VSL of one protomer and S6 of adjacent protomer. These extensive interactions may aid the coupling of conformational changes within the channel domain. One protomer is colour-coded, whereas the adjacent one is coloured silver. b, A stereo view of the polar interaction network between the central domain and CTD. Potential H-bonds are shown as red dashed lines. c, The van der Waals contacts between the U-motif of the central domain and the CTD. Two opposite views are shown. d, Interactions between the central domain and the VSL. Polar and van der Waals contacts are shown on the left and right, respectively.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1. (PDF 160 kb)

Overall structure and domain organization of the RyR1-FKBP12 complex

The animation illustrates the EM density map, the overall structure, and the domain organization of the rabbit RyR1 in complex with FKBP12. (MOV 44971 kb)

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Yan, Z., Bai, Xc., Yan, C. et al. Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution. Nature 517, 50–55 (2015). https://doi.org/10.1038/nature14063

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