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The Central domain of RyR1 is the transducer for long-range allosteric gating of channel opening

The Central domain of RyR1 is the transducer for long-range allosteric gating of channel opening Cell Research (2016) 26:995-1006. www.nature.com/cr ORIGINAL ARTICLE The Central domain of RyR1 is the transducer for long-range allosteric gating of channel opening 1, * 2, 3, 4, * 2, 3, 4, * 2, 3, 4 2, 3, 4 Xiao-Chen Bai , Zhen Yan , Jianping Wu , Zhangqiang Li , Nieng Yan 1 2 MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK; State Key Laboratory of 3 4 Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China 2+ The ryanodine receptors (RyRs) are intracellular calcium channels responsible for rapid release of Ca from the sarcoplasmic/endoplasmic reticulum (SR/ER) to the cytoplasm, which is essential for the excitation-contraction (E-C) coupling of cardiac and skeletal muscles. The near-atomic resolution structure of closed RyR1 revealed the molecular details of this colossal channel, while the long-range allosteric gating mechanism awaits elucidation. Here, we report the cryo-EM structures of rabbit RyR1 in three closed conformations at about 4 Å resolution and an open state at 5.7 Å. Comparison of the closed RyR1 structures shows a breathing motion of the cytoplasmic platform, while the channel domain and its contiguous Central domain remain nearly unchanged. Comparison of the open and closed structures shows a dilation of the S6 tetrahelical bundle at the cytoplasmic gate that leads to channel opening. During the pore opening, the cytoplasmic “O-ring” motif of the channel domain and the U-motif of the Central domain exhibit coupled motion, while the Central domain undergoes domain-wise displacement. These structural analyses provide important insight into the E-C coupling in skeletal muscles and identify the Central domain as the transducer that couples the conformational changes of the cytoplasmic platform to the gating of the central pore. Keywords: RyR1; calcium channel; excitation-contraction coupling; membrane transport; voltage-gated calcium channels Cell Research (2016) 26:995-1006. doi:10.1038/cr.2016.89; published online 29 Jul 2016 Introduction has a mushroom-shaped contour with a square canopy of 270 Å by 270 Å and a height of 160 Å [10-14]. The Ryanodine receptors (RyRs) are responsible for rapid gigantic cytoplasmic region provides the docking station 2+ release of Ca ions from the sarcoplasmic/endoplasmic for a variety of regulators including small molecules, reticulum (SR/ER) to the cytoplasm, a critical step in proteins, and post-translational modifications exempli- the excitation-contraction (E-C) coupling of skeletal and fied by phosphorylation [15, 16]. The identified modu - 2+ cardiac muscles [1-4]. There are three RyR isoforms lators include, but are not limited to Ca , caffeine, ATP, (RyR1-3) in mammals, among which RyR1 is primarily ryanodine, 2,2′,3,5′,6-pentachlorobiphenyl (PCB95), expressed in skeletal muscles, RyR2 mainly functions in and FK506-binding proteins (FKBPs) [17-22]. Among cardiac muscles, and RyR3 remains poorly characterized these, PCB95 was reported to stabilize the fully open [5-9]. state of RyR1 in single-channel recording and [ H]-ryan- The homotetrameric RyRs are the largest known ion odine-binding assay [14]. channels, with a molecular weight of > 2.2 MDa [2, 3]. We recently determined the cryo-EM structure of Electron microscopic examinations showed that RyR1 RyR1 in a closed state with an overall resolution of 3.8 Å [23]. The near-atomic resolution structure resolves 70 % of the 2.2 MDa molecular mass of RyR1 and provides the basis for detailed function-structure correlation analysis. *These three authors contributed equally to this work. a b Correspondence: Nieng Yan , Zhen Yan In addition to the channel domain that exhibits a volt- E-mail: [email protected] age-gated ion channel superfamily fold, nine distinct do- E-mail: [email protected] mains in the cytoplasmic region were identified in each Received 30 May 2016; revised 6 July 2016; accepted 6 July 2016; pub- protomer, including the N-terminal domain (NTD), three lished online 29 Jul 2016 Cryo-EM structures of RyR1 in multiple conformations SPRY domains, the phosphorylation hotspots P1 and P2 reveals a “breathing motion” of the gigantic cytoplasmic domains, the Handle domain, the Helical domain, and scaffold. We will focus on these two conformers, which the Central domain (Supplementary information, Figure display the largest degree of structural shifts, for detailed S1A) [23]. The scaffold of the cytoplasmic region in each analysis. protomer comprises two super spirals, one formed by When the structures of C1 and C4 tetramers are super- the Helical domain, while the other constituted together imposed relative to the channel domain, distinct motions by the armadillo repeats (or the α-solenoid repeats) in of the cytoplasmic domains were observed (Figure 2A). the NTD, the Handle domain, and the Central domain As seen in the morph (Supplementary information, [24]. The plasticity of the cytoplasmic super spirals may Movie S1), the outskirt of the Handle domain and the provide the molecular basis for allosteric gating of the Helical domain, which constitute the corona, appears to pore domain upon stimuli. This hypothesis was in part rock towards the lumen (Figure 2A and 2B). The mo- supported by comparing the cryo-EM structures of RyR1 tion of the NTD is more complex with domains A and B captured in multiple conformations [25]. Nevertheless, moving upwards, while the armadillo repeats-containing the low resolutions prevented reliable definition of the domain C, which immediately precedes the Handle do- channel state. main in the super spiral, moves concordantly with the Here, we report the cryo-EM structures of closed shifts of the corona toward the lumen (Figure 2C and RyR1 between 3.8-4.2 Å resolutions with the cytoplas- Supplementary information, Movie S1). In contrast, mic region exhibiting multiple conformations and an the channel domain and Central domain remain nearly open-state structure at 5.7 Å resolution. Structural com- unchanged (Figure 2D and 2E). Consequently, the coro- parison reveals important insight into the gating mecha- na and NTD appear to pivot around the Central domain nism of RyRs. in each protomer (Supplementary information, Movie S1). It is noteworthy that despite the pronounced struc- tural shifts, there is little intra-domain rearrangement Results when the individual domains in the two structures are “Breathing motion” of the cytoplasmic region of closed compared, suggesting rigid-body shifts of these domains RyR1 that lead to the overall breathing motion of the cytoplas- The RyR1-FKBP12 complex was purified follow- mic region (Supplementary information, Figure S3). ing the previously reported protocol [23]. To acquire a structure in an open state, we tried distinct conditions for Structural determination of RyR1 in the open state sample preparation. In one trial, the protein purified in The structural observation that RyR1 remains closed in 2+ the presence of 0.015% (w/v) Tween-20 was incubated the presence of 10 µM PCB95 and 50 µM Ca was un- with 50 µM CaCl and 10 µM PCB95 before loading to expected. We reasoned that the choice of detergent may the grids for cryo sample preparation. Images were taken also affect the open probability of the channel. Indeed, it on a FEI Tecnai Polara electron microscope operating at was shown that the highest [ H]-ryanodine-binding affin- 300 kV and mounted with a prototype FEI Falcon-III de- ity was achieved in the presence of CHAPS among the tector. Out of 334 000 good particles, three major classes tested detergents, suggesting RyR1 may have a higher were obtained at 3.8 Å, 4.0 Å, and 4.2 Å resolutions open probability when purified in CHAPS [2]. Therefore, (Supplementary information, Figures S1 and S2). we replaced Tween-20 by CHAPS for purification of the The cytoplasmic region of these three classes exhibits RyR1-FKBP12 complex. After data collection and care- gradual shifts (Figure 1A). Unexpectedly, despite the ful classifications, the complex obtained in 0.5% (w/v) 2+ 2+ presence of 50 µM Ca and 10 µM PCB95, the central CHAPS, 10 µM PCB95, and 50 µM Ca gave rise to a pore remains closed in all three classes (Figure 1B). much higher percentage of particles in the open state. Fi- The three new maps all deviate from the published one nally, we were able to reconstruct an EM map, in which in the cytoplasmic region [23] (Figure 1A). The cytoplas- the central pore appeared open, with an overall resolution mic region, particularly the previously defined corona of 5.7 Å (Supplementary information, Figures S1 and peripheral zones, in the four conformers displays a and S2). Despite that the side chains were not traceable consecutive conformational transition (Supplementary at this moderate resolution, most of the secondary struc- information, Movie S1). We name the four conformers tures can be reliably assigned based on the near-atomic C1 through C4, among which C2 is the published one structure of the closed RyR1 (Figure 3A). [23]. From C1 to C4, the corona of the cytoplasmic re- Comparing the new map to the four closed RyR1 maps gion moves gradually toward the lumen (Figure 1A). The shows that the overall conformation of the cytoplasmic morph generated based on the conformers C1 and C4 region in the new map closely resembles that of C3 con- SPRINGER NATURE | Cell Research | Vol 26 No 9 | September 2016 Xiao-Chen Bai et al. Figure 1 Cryo-EM structures of RyR1 in three additional closed conformations. (A) Three additional RyR1 structures were obtained. Compared with the previously reported RyR1 structure, the cytoplasmic region of the three new classes undergoes pronounced shifts. The four classes of closed RyR1 are designated C1 through C4, among which C2 is the previously report- ed one [23]. Shown here are the superimpositions of the three new structures with the C2 conformer. The arrows indicate the structural shifts from C2 to the indicated conformer. Please refer to Supplementary information, Movie S1 for the confor- mational transition from C1 to C4. (B) The channel domain remains nearly identical in the four classes. Shown here are the cytoplasmic views of the pore-forming segments. The EM maps were generated in Chimera [47]. former (Supplementary information, Figure S4). To NTD (Supplementary information, Figure S5). In pinpoint the switch for pore opening, we focus on the contrast, superimposition of EM maps of the channel do- comparison between the new map and that of the C3 main reveals obvious dilation of the pore, supporting the conformer. Similar to the aforementioned comparison be- definition of an open channel ( Figure 3B). The channel tween C1 and C4 conformers, when individual domains domain, including the pore-forming S5 and S6 segments in the cytoplasmic region are compared, little change is and the voltage-sensor like (VSL) domain, undergoes an found within the Helical domain, Handle domain and overall small-degree counterclockwise rotation from the www.cell-research.com | Cell Research | SPRINGER NATURE Cryo-EM structures of RyR1 in multiple conformations closed to open state when viewed from the cytoplasmic On the other side, the transmembrane fragment close side (Figure 3B). to the cytoplasm and cytoplasmic extension of the S6 segment (designated the S6 segment hereafter) swing Cyt Cryo-EM structure of the open RyR1 outwards, resulting in the dilation of the intracellular gate The moderate resolution of the open RyR1 structure (Figure 4A, Supplementary information, Movie S2). disallowed side chain assignment. Nevertheless, the The distance between the Cα atoms of the constriction pronounced backbone shifts of S6 segments support the site residue Ile4937 in the diagonal protomers increases open conformation of the new structure as seen in the from 10.4 Å to 15.6 Å (Figure 4B). The calculated pore EM map (Figure 3B, Supplementary information, diameter of the constriction site in the high-resolution 2+ Figure S2B). When the structures of the open- and closed RyR1 was ~1.6 Å, blocking Ca passage. Now closed (C3)-RyR1 are overlaid relative to the channel that the diameter is expanded by ~5 Å, it would allow 2+ domain, the luminal halves including those of S5, S6, the permeation of single-file Ca even with hydration shell selectivity filter, and P loops, remain nearly unchanged. (Supplementary information, Movie S2). Figure 2 Conformational changes of the individual domains between C1 and C4 conformers. (A) Structural comparison be- tween C1 (gray) and C4 (violet) protomers. The two tetrameric structures are superimposed relative to the channel domain. The violet arrows indicate the conformational changes from C1 to C4. The same is applied to the other panels. (B) Structural shifts of the corona between C1 and C4 tetramers. The outskirt of the corona, composed of the Handle and Helical domains, moves towards the SR lumen from C1 to C4. Shown here is a side view. (C) The NTD undergoes a rocking motion with the central domains A and B upwards and the outer domain C downwards. For visual clarity, only two diagonal protomers are shown in side view. (D, E) The Central (D) and channel (E) domains remain nearly unchanged between C1 and C4 states. All structure figures were prepared with PyMol [48]. SPRINGER NATURE | Cell Research | Vol 26 No 9 | September 2016 Xiao-Chen Bai et al. To identify the deviation point of the S6 segments, the two structures are superimposed relative to the selectivity filter (SF) and the supporting helices (residues 4 836 to 4 935; Figure 4C). An ~15° bending of S6 helix occurs at a conserved Gly residue (Gly4934 in RyR1; Figure 4C). The structural observation supports a recent characterization that substitution of Gly4934 led to altered channel gating and ion conductance. Gly may provide the molecular basis for conformational flexibility [26]. Coupled conformational changes of the cytoplasmic O-ring of the channel domain As analyzed previously, the S6 segment, CTD, and Cyt the cytoplasmic segments of the VSL domain (designated the VSL domain) together constitute an O-ring (Figure Cyt 5A). When the channel domains in the structures of the open and C3 states are superimposed relative to the SF and the supporting helices, the elements in the cytoplas- Figure 3 The cryo-EM reconstruction of an open RyR1. (A) The mic O-ring appear to undergo concordant shift (Figures overall cryo-EM map of the open RyR1 at 5.7 Å resolution. Two 3B and 5A). Comparison of the individual domains perpendicular views are shown. (B) Comparison of the channel shows little intra-domain rearrangement within the VSL domain between the open and the C3 conformers. Shown on the right is the cytoplasmic view. The arrows indicate the confor- mational change from the C3 (blue) to the open (yellow) state. Figure 4 Dilation of the inner gate leads to channel opening. (A) The cytoplasmic gate of the S6 bundle undergoes a dilation that leads to pore opening, while the luminal segments have little change. (B) Dilation of the cytoplasmic gate. The indicated distances are measured between the Cα atoms of the gating residue Ile4937 on the S6 segments in the diagonal protomers. Please refer to Supplementary information, Movie S2 for the conformational changes of the channel domain. (C) Com- parison of the S6 segment relative to the luminal halves of the pore-forming segments (residues 4 836-4 935) shows that the structural deviation between the open (yellow) and closed (blue) states occurs at Gly4934. www.cell-research.com | Cell Research | SPRINGER NATURE Cryo-EM structures of RyR1 in multiple conformations or CTD domain (Figure 5B and 5C). In addition, there is and Ile4936 on S6 in the same protomer also remain no relative motion between the CTD and the C-terminal nearly the same in the open and C3 structures (Figure segment of S6, supporting our previous finding that the 5D, lower panel; Supplementary information, Movie presence of a zinc-finger motif at the joint of CTD and S3). S6 may rigidify the two structural moieties [23] (Figure The structural observations suggest that shifts of VSL 5C). and CTD may pull the S6 segments as well as the con- The extensive interactions between the S5 and S6 straining S4-5 segments outwards to open the intracellu- segments within one protomer and those between the lar gate. VSL and the pore-forming segments in the neighbouring protomer were analyzed in details in our previous report Coupled conformational changes between the cytoplas- [23]. We predicted that these extensive interactions may mic O-ring of the channel domain and the U-motif of the provide the molecular basis for coupled conformational Central domain changes. Indeed, comparison of the open and C3 struc- As shown previously, the cytoplasmic O-ring of the tures shows that these elements undergo coupled mo- channel domain accommodates the U-motif in the Cen- tion during pore opening (Figure 5D, Supplementary tral domain. The helical hairpin of the U-motif pierces information, Movie S3). For instance, the inter-helical through the O-ring, whereas the anti-parallel β-strands distances between S4 in one protomer and S5 in the ad- are located at the concave surface below the O-ring (Fig- jacent protomer in the C3 structure are similar to those ure 6A). The intricate interactions between the U-motif in the open structure (Figure 5D, upper panels). Simi- and the O-ring tie them into a stable unit. Indeed, the larly, the distances between the Cα atoms of Ile4826 on U-motif moves together with the O-ring during pore the S4-5 segment and Ile4931 on the S6 segment in the opening (Figure 6A). When the open and C3 structures neighbouring protomers and between Val4830 on S4-5 are superimposed relative to CTD, the U-motif can be Figure 5 Coupled conformational shifts of the segments within the channel domain during pore opening. (A) Structural com- parison of the channel domain in one protomer between the open and C3 structures relative to the luminal halves of the pore-forming segments (residues 4 836-4 935). The cytoplasmic “O-ring” composed of the cytoplasmic segments of S6 (S6 ), Cyt the VSL , and the CTD appears to undergo concordant shifts. (B) The VSL appears rigid during pore opening. There is no Cyt intra-domain rearrangement observed between the VSL domain in the open and closed structures. Therefore, the VSL do- main undergoes a rigid-body shift during pore opening. (C) There is no relative motion between CTD and the S6 segment Cyt during the pore dilation. (D) The concerted motions between the S5 and S6 segments in the same protomer, and between the S5 segment and the S4 and S4-5 segments in the neighbouring protomers. The distances between the C atoms of the indicated residues are presented in Å. For visual clarity, the specified protomer is coloured blue and yellow in the closed and open states, respectively, while the neighbouring protomer is coloured green in both states. The other two protomers that are not discussed are coloured gray. Please refer to Supplementary information, Movie S3 for the concordant shifts of the channel segments during pore dilation. SPRINGER NATURE | Cell Research | Vol 26 No 9 | September 2016 Xiao-Chen Bai et al. Figure 6 Coupled conformational changes between the channel domain and the Central domain. (A) Concordant confor- mational changes between the cytoplasmic “O-ring” of the channel domain and the U-motif of the Central domain. Shown here are the luminal and side views of the U-motif and the O-ring. The U-motif in the open RyR1 is coloured orange. Please refer to Supplementary information, Movie S4 for the concerted conformational changes. (B) Structural comparison of the O-ring and the U-motif between the C3 conformer and the open structure relative to CTD. There is little shift of the U-motif relative to CTD. (C) Comparison of the Central domain between the open and the C3 structures relative to the tetramer- ic channel domain (left panel) and relative to the individual Central domain (right panel). The tetrameric Central domain is shown in cytoplasmic view. The yellow and green arrows indicate the conformational transition from C3 to the open state. almost completely overlaid, indicating coupled motion may lead to the U-motif shift. between the CTD and the U-motif (Figure 6B). The Central domain undergoes a slight outward dis- The above analysis is based on the structure deviation placement from the closed to open conformation when between the open- and closed-RyR1 structures. In terms viewed from the SR lumen (Figure 6C). Within the Cen- of gating, the channel is pulled-open by signals applied tral domain, the U-motif and the nearby helix α20, as to the cytoplasmic region. Therefore, it is likely that the well as the extruding helix α4, slightly squeeze toward displacement of U-motif mobilizes the O-ring, leading the center of the concave side of the armadillo repeats, to the dilation of the S6 segments. We next analyzed the but there is little rearrangement of the armadillo repeats conformational changes of the cytoplasmic domains that (Figure 6C, right panel). The domainwise shift and the www.cell-research.com | Cell Research | SPRINGER NATURE Cryo-EM structures of RyR1 in multiple conformations intra-domain rearrangements of the Central domain like- surface of the Central domain and the amino terminal ly provide the pulling-force for the channel domain. We helices α1a and α1b in the Helical domain (Figure 7A). then examined the potential effect of the corona and pe- Similarly, the NTD also slightly revolves around the ripheral domains on the structural changes of the Central interface with the Central domain, but to the opposite domain. direction of the rotation of the Helical domain relative to the Central domain (Figure 7B). As the Handle domain Lateral rotation of the Central domain triggered by and the armadillo repeats of the NTD are consecutive, it structural shifts of the NTD, Handle and Helical domains is not surprising that the rotation of the Handle domain To understand the potential action of other cytoplas- is consistent with that of the NTD, i.e., centering around mic domains on the Central domain, we compared them the interface with the Central domain (Figure 7C). pairwise between the open and C3 structures (Figure 7A- In our previous analysis of the 3.8 Å structure of 7C, Supplementary information, Movie S4). When closed RyR1, we paid particular attention to the inter- the Central and Helical domains are compared relative action network among the super spiral assemblies in with the Central domain, the superspiral of the Helical the cytoplasmic region. Basically, all the armadillo re- domain appears to rotate around the interface between peats-containing domains contact each other (Figure 7D- the two domains involving the amino terminal concave 7F). They together constitute a network of pronounced Figure 7 The extensive interaction network among the cytoplasmic domains provide the molecular basis for long-range al- losteric gating. (A-C) The motions of the Helical domain, NTD, and Handle domain relative to the Central domain between the closed and open structures. The comparison is made relative to the Central domain (labeled with asterisk). In all panels, domains in C3 are blue, while those in the open structure are domain-coloured. (D) Extensive interfaces among the armadillo repeats-containing cytoplasmic domains, including the NTD, Helical, Handle, and Central domains. The NTD from the neigh- bouring protomer is also shown, coloured pale yellow and labeled NTD’. (E) The extensive internal interactions within one cytoplasmic superhelical assembly consisting of the armadillo repeats in NTD, the Handle domain, and the Central domain. Note that the Central domain also interacts with the NTD. (F) The extensive interaction network involving the Helical domain, the Handle domain, the Central domain in one protomer and the NTD in the adjacent protomer. Please refer to our previous publication [23] for detailed analysis of the cytoplasmic interaction network. SPRINGER NATURE | Cell Research | Vol 26 No 9 | September 2016 Xiao-Chen Bai et al. plasticity, which can transduce the conformational chang- structures and between the closed and open structures es initiated to any point of the helical surface. The col- are compared (Supplementary information, Movies lective motions of the Helical domain, the NTD, and the S1 and S4). It is evident that the corona, peripheral do- Handle domain may lead to the observed compression of mains, and NTD of the cytoplasmic region undergo ver- the Central domain toward its concave side (Figure 6C, tical motions during the conformational changes between right panel). In addition to the internal rearrangement of the distinct closed states (Supplementary information, the Central domain, an overall concerted lateral rotation Movie S1). In contrast, despite the overall similarity of the Central and the other cytoplasmic domains occur between the open and C3 structures, the lateral rotation between the open and closed states when viewed from of the cytoplasmic domains is evident (Supplementary the side (Supplementary information, Movie S4). information, Movie S4). As illustrated above, the pore opening requires dilation Discussion of the S6 helical bundle at the intracellular gate (Figure 4). The shift of S6 is triggered by the motion of VSL and Cyt In this study, we report the structures of RyR1 in three CTD (Figure 5), which is induced by the displacement of closed states and an open state. It is particularly interest- the U-motif in the Central domain (Figure 6). The shift ing when the conformational changes among the closed of the U-motif results from both intra-domain rearrange- Figure 8 Speculative mechanism of the excitation-contraction coupling. (A) Conformational changes to any cytoplasmic domain may be propagated to the Central domain along the interaction network described in Figure 7. Shown here are side views of tetrameric RyR1. Inset: an example of a speculative route (black arrows) for the propagation of conformational changes that can be triggered by motion of the SPRY3 domain. (B) Speculative model of the complex between RyR1 and the Ca 1.1 complex. Structural determination of both RyR1 and the Ca 1.1 complex provides the foundation for elucidating the v v molecular mechanism of RyR1 opening induced by depolarization of the plasma membrane. Structures of the Ca 1.1 com- plex (PDB code: 3JBR) [34] and RyR1 were manually docked in COOT [43]. www.cell-research.com | Cell Research | SPRINGER NATURE Cryo-EM structures of RyR1 in multiple conformations The fractions containing RyR1-FKBP12 complex were pooled for ment and overall lateral rotation of the Central domain. EM analysis. Before loading to grids for cryo sample preparation, In essence, the pore opening requires mobilization of the the complex was incubated with 50 µM CaCl and 10 µM PCB95 Central domain, which thereby serves as the transducer for 30 min on ice. The sample that gave rise to the open struc- of the long-range conformational changes. ture was purified with 0.5% CHAPS (w/v; Amresco) instead of Under physiological condition, RyR1 is activated Tween-20. The other procedures were the same. through direct physical contacts with the Ca 1.1 complex as well as the surrounding RyR1 tetramers in the crystal- Cryo-EM image acquisition Aliquots of 3 µl purified RYR1 at a concentration of ~30 nM line-like assembly [27-32]. It was shown that multiple ar- were placed on glow-discharged holey carbon grids (Quantifoil eas of the RyR1 cytoplasmic region, such as the SPRY3 Cu, R2/2), on which a home-made continuous carbon film (esti - domain, are involved in the coupling with Cav1.1 com- mated to be ~30 Å thick) had previously been deposited. Grids plex (Figure 8A) [33]. Note that the SPRY3 domain is in were blotted for 2 s and flash-frozen in liquid ethane using an FEI direct contact with NTD. Potential shifts of the SPRY3 Vitrobot II. Grids were transferred to an FEI Tecnai Polara electron domain may be translated to the conformational changes microscope that was operating at 300 kV. Images were recorded of NTD, and subsequently the Handle domain and the manually using a prototype FEI Falcon-III detector at a calibrated Central domain. We speculate that the shift of SPRY3 magnification of 104 478, yielding a pixel size of 1.34 Å. A dose rate of 20 electrons/Å /s, and an exposure time of 2 s were used on may involve a displacement to trigger the lateral rotation the Falcon. of the cytoplasmic domains of RyR1 (Figure 8A, inset). Although the elements in the Ca 1.1 complex that bind Image processing to RyR1 remain to be elucidated, the recent structural de- Similar image processing procedures were employed as report- termination of the Ca 1.1 complex laid out the foundation ed [23]. We used MOTIONCORR [35] for whole-frame motion for investigation of the gating mechanism of RyR1 (Fig- correction, CTFFIND3 [36] for estimation of the contrast transfer ure 8B) [34]. Structures of the complex between Ca 1.1 function parameters, and RELION-1.4 [37] for all subsequent steps. References for template-based particle picking [38] were and RyR1 as well as the structures of Ca 1.1 in multiple obtained from 2D class averages that were calculated from a man- states would offer the answer to address the fundamental ually picked subset of the micrographs. A 20 Å low-pass filter was problem of how depolarization of the plasma membrane applied to these templates to limit model bias. To discard false pos- would induce the pore opening of RyR1, which resides in itives from the picking, we used initial runs of 2D and 3D classifi- the SR membrane. We speculate that the conformation- cation to remove bad particles from the data. The selected particles al changes of the voltage-sensing domains of Ca 1.1α1 v were then submitted to 3D auto-refinement, particle-based motion upon depolarization would induce shifts of the β-subunit correction and radiation-damage weighting [38]. The resulting “polished particles” were used for masked classification only on and other cytoplasmic segments of Ca 1.1, which may pore region with subtraction of the residual signal [35], and the trigger the motion of the adjoining RyR1 cytoplasmic do- original particle images from the resulting classes were submitted mains exemplified by the SPRY3 domain. The structural to a second round of 3D auto-refinement. All 3D classifications shifts at the periphery of the RyR1 cytoplasmic region and 3D refinements were started from a 40 Å low-pass filtered ver - are propagated along the superhelical assemblies of the sion of the high-resolution consensus structure. Fourier Shell Co- cytoplasmic domains to the Central domain, eventual- efficient (FSC) curves were corrected for the effects of a soft mask ly leading to the opening of the intracellular gate. The on the FSC curve using high-resolution noise substitution [39]. speculative mechanism awaits experimental evidence. Reported resolutions are based on gold-standard refinement pro- cedures and the corresponding FSC = 0.143 criterion [40]. Prior to In addition, high resolution structures of RyR channels visualization, all density maps were corrected for the modulation in various states are required to reveal the modulation of 2+ transfer function of the detector, and then sharpened by applying a the channel activity by multiple signals such as Ca and negative B-factor that was estimated using automated procedures PCB95. [41]. 2+ For the sample purified in TWEEN-20/PCB95/Ca , 334K Materials and Methods particles were selected after initial 2D and 3D classification. Subsequent 3D auto-refinement and particle polishing yielded a map with relatively fuzzy densities in the cytoplasmic region. 3D Protein purification The RyR1-FKBP12 complex that was captured in multiple classification into five classes with small angular sampling yielded closed conformations was purified following similar protocol as three classes with better density of the cytoplasmic region. Rel- before with slight modifications [23]. The buffer for the last step atively poor reconstructed density was observed in the other two size-exclusion chromatography (Superdex-200, 10/30, GE Health- classes. Separate 3D auto-refinements of the corresponding parti - care) purification was changed to 20 mM MOPS-Na, pH 7.4, 250 cles in the original data set for the three best classes gave rise to mM NaCl, 2 mM DTT, 0.015% Tween-20 (w/v; Sigma-Aldrich) reconstructions to 3.8-4.2 Å resolution (also see Supplementary and protease inhibitor cocktail including 2 mM PMSF, 2.6 µg/ml information, Figure S1 and Table S1). 2+ aprotinin, 1.4 µg/ml pepstatin, and 10 µg/ml leupeptin (Amresco). For the sample purified in CHAPS/PCB95/Ca , initial classifi- SPRINGER NATURE | Cell Research | Vol 26 No 9 | September 2016 Xiao-Chen Bai et al. cation selected 46K particles. After particle polishing, application tion in calcium release. Cold Spring Harb Perspect Biol 2010; of the masked classification procedure on the pore region with 2:a003996. residual signal subtraction into three classes identified a single 5 Takeshima H, Nishimura S, Matsumoto T, et al. Primary class with good density and open conformation. After 3D auto-re- structure and expression from complementary DNA of skele- finement, the corresponding 30K particles gave a map with a reso- tal muscle ryanodine receptor. Nature 1989; 339:439-445. lution of 5.7 Å. 6 Rossi D, Sorrentino V. Molecular genetics of ryanodine recep- tors Ca2+-release channels. Cell Calcium 2002; 32:307-319. 7 Otsu K, Willard HF, Khanna VK, Zorzato F, Green NM, Ma- Model building and refinement The initial model (PDB: 3J8H) was docked into each map by cLennan DH. Molecular cloning of cDNA encoding the Ca2+ using DireX [42]. The resulting models were manually adjusted release channel (ryanodine receptor) of rabbit cardiac muscle in COOT [43] to further improve the fitting of secondary struc - sarcoplasmic reticulum. J Biol Chem 1990; 265:13472-13483. tures and side chains. Subsequently, all models were refined using 8 Nakai J, Imagawa T, Hakamat Y, Shigekawa M, Takeshima REFMAC [44] with secondary structure restraints generated by H, Numa S. Primary structure and functional expression from ProSMART [45]. To prevent overfitting, the optimal weight for cDNA of the cardiac ryanodine receptor/calcium release chan- nel. FEBS Lett 1990; 271:169-177. refinement in REFMAC were determined by cross-validation [46]. 9 Hakamata Y, Nakai J, Takeshima H, Imoto K. Primary struc- ture and distribution of a novel ryanodine receptor/calcium Accession codes The atomic coordinates of the C1, C3, C4, and open-RyR1 release channel from rabbit brain. FEBS Lett 1992; 312:229- structures have been deposited in the Protein Data Bank with the 235. accession codes 5GKY, 5GKZ, 5GL0, and 5GL1, respectively. The 10 Radermacher M, Wagenknecht T, Grassucci R, et al. Cryo- cryo-EM maps have been deposited to EMDB with the following EM of the native structure of the calcium release channel/ accession codes: EMD-9518 (C1), EMD-9519 (C3), EMD-9520 ryanodine receptor from sarcoplasmic reticulum. Biophys J (C4), and EMD-9521 (open state). 1992; 61:936-940. 11 Radermacher M, Rao V, Grassucci R, et al. Cryo-electron mi- croscopy and three-dimensional reconstruction of the calcium Acknowledgments release channel/ryanodine receptor from skeletal muscle. J We thank the Tsinghua University Branch of China National Cell Biol 1994; 127:411-423. Center for Protein Sciences (Beijing) for providing the facility 12 Samso M, Wagenknecht T, Allen PD. Internal structure and support. This work was supported by the Ministry of Science and visualization of transmembrane domains of the RyR1 calci- Technology of China (2015CB9101012014 and ZX09507003006), um release channel by cryo-EM. Nat Struct Mol Biol 2005; and the National Natural Science Foundation of China (31321062 12:539-544. and 81590761). The research of Nieng Yan was supported in part 13 Serysheva, II, Ludtke SJ, Baker ML, et al. Subnanometer-res- by an International Early Career Scientist grant from the Howard olution electron cryomicroscopy-based domain models for the Hughes Medical Institute and an endowed professorship from cytoplasmic region of skeletal muscle RyR channel. Proc Natl Bayer Healthcare. Acad Sci USA 2008; 105:9610-9615. 14 Samso M, Feng W, Pessah IN, Allen PD. Coordinated move- ment of cytoplasmic and transmembrane domains of RyR1 Author Contributions NY conceived the project. ZY, XB, JW, and NY designed upon gating. PLoS Biol 2009; 7:e85. experiments. XB, ZY, JW, and ZL performed experiments. All au- 15 Rodriguez P, Bhogal MS, Colyer J. Stoichiometric phosphor- thors analyzed the data and contributed to manuscript preparation. ylation of cardiac ryanodine receptor on serine 2809 by calm- NY and ZY wrote the manuscript. odulin-dependent kinase II and protein kinase A. J Biol Chem 2003; 278:38593-38600. 16 Wehrens XH, Lehnart SE, Reiken SR, Marks AR. 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Regulation in RELION-1.3. J Struct Biol 2015; 189:114-122. of ryanodine receptors by FK506 binding proteins. Trends 39 Chen S, McMullan G, Faruqi AR, et al. High-resolution noise Cardiovasc Med 2004; 14:227-234. substitution to measure overfitting and validate resolution in 23 Yan Z, Bai XC, Yan C, et al. Structure of the rabbit ryano- 3D structure determination by single particle electron cryomi- dine receptor RyR1 at near-atomic resolution. Nature 2015; croscopy. Ultramicroscopy 2013; 135:24-35. 517:50-55. 40 Scheres SH, Chen S. Prevention of overfitting in cryo-EM 24 Tewari R, Bailes E, Bunting KA, Coates JC. Armadillo-repeat structure determination. Nat Methods 2012; 9:853-854. protein functions: questions for little creatures. Trends Cell 41 Rosenthal PB, Henderson R. Optimal determination of parti- Biol 2010; 20:470-481. cle orientation, absolute hand, and contrast loss in single-par- 25 Efremov RG, Leitner A, Aebersold R, Raunser S. Architec- ticle electron cryomicroscopy. 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Re- Acta Crystallogr D Biol Crystallogr 1997; 53:240-255. gions of the skeletal muscle dihydropyridine receptor critical 45 Nicholls RA, Fischer M, McNicholas S, Murshudov GN. for excitation-contraction coupling. Nature 1990; 346:567- Conformation-independent structural comparison of macro- 569. molecules with ProSMART. Acta Crystallogr D Biol Crystal- 29 Franzini-Armstrong C, Protasi F, Ramesh V. Shape, size, and logr 2014; 70:2487-2499. distribution of Ca(2+) release units and couplons in skeletal 46 Fernandez IS, Bai XC, Murshudov G, Scheres SH, Ra- and cardiac muscles. Biophys J 1999; 77:1528-1539. makrishnan V. Initiation of translation by cricket paralysis vi- 30 Protasi F, Franzini-Armstrong C, Flucher BE. Coordinated in- rus IRES requires its translocation in the ribosome. Cell 2014; corporation of skeletal muscle dihydropyridine receptors and 157:823-831. ryanodine receptors in peripheral couplings of BC3H1 cells. J 47 Pettersen EF, Goddard TD, Huang CC, et al. 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Structure of the voltage-gated calci- transmit the Contribution as long as it attributed back um channel Cav1.1 complex. Science 2015; 350:aad2395. to the author. Readers are permitted to alter, transform 35 Bai XC, Rajendra E, Yang G, Shi Y, Scheres SH. Sampling or build upon the Contribution as long as the resulting work is then the conformational space of the catalytic subunit of human distributed under this is a similar license. Readers are not permitted gamma-secretase. eLife 2015; 4:e11182. to use the Contribution for commercial purposes. Please read the full 36 Mindell JA, Grigorieff N. Accurate determination of local de- license for further details at - http://creativecommons.org/ licenses/by- focus and specimen tilt in electron microscopy. J Struct Biol nc-sa/4.0/ 2003; 142:334-347. 37 Scheres SH. RELION: implementation of a Bayesian ap- © The Author(s) 2016 proach to cryo-EM structure determination. 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The Central domain of RyR1 is the transducer for long-range allosteric gating of channel opening

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Life Sciences; Life Sciences, general; Cell Biology
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Abstract

Cell Research (2016) 26:995-1006. www.nature.com/cr ORIGINAL ARTICLE The Central domain of RyR1 is the transducer for long-range allosteric gating of channel opening 1, * 2, 3, 4, * 2, 3, 4, * 2, 3, 4 2, 3, 4 Xiao-Chen Bai , Zhen Yan , Jianping Wu , Zhangqiang Li , Nieng Yan 1 2 MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK; State Key Laboratory of 3 4 Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China 2+ The ryanodine receptors (RyRs) are intracellular calcium channels responsible for rapid release of Ca from the sarcoplasmic/endoplasmic reticulum (SR/ER) to the cytoplasm, which is essential for the excitation-contraction (E-C) coupling of cardiac and skeletal muscles. The near-atomic resolution structure of closed RyR1 revealed the molecular details of this colossal channel, while the long-range allosteric gating mechanism awaits elucidation. Here, we report the cryo-EM structures of rabbit RyR1 in three closed conformations at about 4 Å resolution and an open state at 5.7 Å. Comparison of the closed RyR1 structures shows a breathing motion of the cytoplasmic platform, while the channel domain and its contiguous Central domain remain nearly unchanged. Comparison of the open and closed structures shows a dilation of the S6 tetrahelical bundle at the cytoplasmic gate that leads to channel opening. During the pore opening, the cytoplasmic “O-ring” motif of the channel domain and the U-motif of the Central domain exhibit coupled motion, while the Central domain undergoes domain-wise displacement. These structural analyses provide important insight into the E-C coupling in skeletal muscles and identify the Central domain as the transducer that couples the conformational changes of the cytoplasmic platform to the gating of the central pore. Keywords: RyR1; calcium channel; excitation-contraction coupling; membrane transport; voltage-gated calcium channels Cell Research (2016) 26:995-1006. doi:10.1038/cr.2016.89; published online 29 Jul 2016 Introduction has a mushroom-shaped contour with a square canopy of 270 Å by 270 Å and a height of 160 Å [10-14]. The Ryanodine receptors (RyRs) are responsible for rapid gigantic cytoplasmic region provides the docking station 2+ release of Ca ions from the sarcoplasmic/endoplasmic for a variety of regulators including small molecules, reticulum (SR/ER) to the cytoplasm, a critical step in proteins, and post-translational modifications exempli- the excitation-contraction (E-C) coupling of skeletal and fied by phosphorylation [15, 16]. The identified modu - 2+ cardiac muscles [1-4]. There are three RyR isoforms lators include, but are not limited to Ca , caffeine, ATP, (RyR1-3) in mammals, among which RyR1 is primarily ryanodine, 2,2′,3,5′,6-pentachlorobiphenyl (PCB95), expressed in skeletal muscles, RyR2 mainly functions in and FK506-binding proteins (FKBPs) [17-22]. Among cardiac muscles, and RyR3 remains poorly characterized these, PCB95 was reported to stabilize the fully open [5-9]. state of RyR1 in single-channel recording and [ H]-ryan- The homotetrameric RyRs are the largest known ion odine-binding assay [14]. channels, with a molecular weight of > 2.2 MDa [2, 3]. We recently determined the cryo-EM structure of Electron microscopic examinations showed that RyR1 RyR1 in a closed state with an overall resolution of 3.8 Å [23]. The near-atomic resolution structure resolves 70 % of the 2.2 MDa molecular mass of RyR1 and provides the basis for detailed function-structure correlation analysis. *These three authors contributed equally to this work. a b Correspondence: Nieng Yan , Zhen Yan In addition to the channel domain that exhibits a volt- E-mail: [email protected] age-gated ion channel superfamily fold, nine distinct do- E-mail: [email protected] mains in the cytoplasmic region were identified in each Received 30 May 2016; revised 6 July 2016; accepted 6 July 2016; pub- protomer, including the N-terminal domain (NTD), three lished online 29 Jul 2016 Cryo-EM structures of RyR1 in multiple conformations SPRY domains, the phosphorylation hotspots P1 and P2 reveals a “breathing motion” of the gigantic cytoplasmic domains, the Handle domain, the Helical domain, and scaffold. We will focus on these two conformers, which the Central domain (Supplementary information, Figure display the largest degree of structural shifts, for detailed S1A) [23]. The scaffold of the cytoplasmic region in each analysis. protomer comprises two super spirals, one formed by When the structures of C1 and C4 tetramers are super- the Helical domain, while the other constituted together imposed relative to the channel domain, distinct motions by the armadillo repeats (or the α-solenoid repeats) in of the cytoplasmic domains were observed (Figure 2A). the NTD, the Handle domain, and the Central domain As seen in the morph (Supplementary information, [24]. The plasticity of the cytoplasmic super spirals may Movie S1), the outskirt of the Handle domain and the provide the molecular basis for allosteric gating of the Helical domain, which constitute the corona, appears to pore domain upon stimuli. This hypothesis was in part rock towards the lumen (Figure 2A and 2B). The mo- supported by comparing the cryo-EM structures of RyR1 tion of the NTD is more complex with domains A and B captured in multiple conformations [25]. Nevertheless, moving upwards, while the armadillo repeats-containing the low resolutions prevented reliable definition of the domain C, which immediately precedes the Handle do- channel state. main in the super spiral, moves concordantly with the Here, we report the cryo-EM structures of closed shifts of the corona toward the lumen (Figure 2C and RyR1 between 3.8-4.2 Å resolutions with the cytoplas- Supplementary information, Movie S1). In contrast, mic region exhibiting multiple conformations and an the channel domain and Central domain remain nearly open-state structure at 5.7 Å resolution. Structural com- unchanged (Figure 2D and 2E). Consequently, the coro- parison reveals important insight into the gating mecha- na and NTD appear to pivot around the Central domain nism of RyRs. in each protomer (Supplementary information, Movie S1). It is noteworthy that despite the pronounced struc- tural shifts, there is little intra-domain rearrangement Results when the individual domains in the two structures are “Breathing motion” of the cytoplasmic region of closed compared, suggesting rigid-body shifts of these domains RyR1 that lead to the overall breathing motion of the cytoplas- The RyR1-FKBP12 complex was purified follow- mic region (Supplementary information, Figure S3). ing the previously reported protocol [23]. To acquire a structure in an open state, we tried distinct conditions for Structural determination of RyR1 in the open state sample preparation. In one trial, the protein purified in The structural observation that RyR1 remains closed in 2+ the presence of 0.015% (w/v) Tween-20 was incubated the presence of 10 µM PCB95 and 50 µM Ca was un- with 50 µM CaCl and 10 µM PCB95 before loading to expected. We reasoned that the choice of detergent may the grids for cryo sample preparation. Images were taken also affect the open probability of the channel. Indeed, it on a FEI Tecnai Polara electron microscope operating at was shown that the highest [ H]-ryanodine-binding affin- 300 kV and mounted with a prototype FEI Falcon-III de- ity was achieved in the presence of CHAPS among the tector. Out of 334 000 good particles, three major classes tested detergents, suggesting RyR1 may have a higher were obtained at 3.8 Å, 4.0 Å, and 4.2 Å resolutions open probability when purified in CHAPS [2]. Therefore, (Supplementary information, Figures S1 and S2). we replaced Tween-20 by CHAPS for purification of the The cytoplasmic region of these three classes exhibits RyR1-FKBP12 complex. After data collection and care- gradual shifts (Figure 1A). Unexpectedly, despite the ful classifications, the complex obtained in 0.5% (w/v) 2+ 2+ presence of 50 µM Ca and 10 µM PCB95, the central CHAPS, 10 µM PCB95, and 50 µM Ca gave rise to a pore remains closed in all three classes (Figure 1B). much higher percentage of particles in the open state. Fi- The three new maps all deviate from the published one nally, we were able to reconstruct an EM map, in which in the cytoplasmic region [23] (Figure 1A). The cytoplas- the central pore appeared open, with an overall resolution mic region, particularly the previously defined corona of 5.7 Å (Supplementary information, Figures S1 and peripheral zones, in the four conformers displays a and S2). Despite that the side chains were not traceable consecutive conformational transition (Supplementary at this moderate resolution, most of the secondary struc- information, Movie S1). We name the four conformers tures can be reliably assigned based on the near-atomic C1 through C4, among which C2 is the published one structure of the closed RyR1 (Figure 3A). [23]. From C1 to C4, the corona of the cytoplasmic re- Comparing the new map to the four closed RyR1 maps gion moves gradually toward the lumen (Figure 1A). The shows that the overall conformation of the cytoplasmic morph generated based on the conformers C1 and C4 region in the new map closely resembles that of C3 con- SPRINGER NATURE | Cell Research | Vol 26 No 9 | September 2016 Xiao-Chen Bai et al. Figure 1 Cryo-EM structures of RyR1 in three additional closed conformations. (A) Three additional RyR1 structures were obtained. Compared with the previously reported RyR1 structure, the cytoplasmic region of the three new classes undergoes pronounced shifts. The four classes of closed RyR1 are designated C1 through C4, among which C2 is the previously report- ed one [23]. Shown here are the superimpositions of the three new structures with the C2 conformer. The arrows indicate the structural shifts from C2 to the indicated conformer. Please refer to Supplementary information, Movie S1 for the confor- mational transition from C1 to C4. (B) The channel domain remains nearly identical in the four classes. Shown here are the cytoplasmic views of the pore-forming segments. The EM maps were generated in Chimera [47]. former (Supplementary information, Figure S4). To NTD (Supplementary information, Figure S5). In pinpoint the switch for pore opening, we focus on the contrast, superimposition of EM maps of the channel do- comparison between the new map and that of the C3 main reveals obvious dilation of the pore, supporting the conformer. Similar to the aforementioned comparison be- definition of an open channel ( Figure 3B). The channel tween C1 and C4 conformers, when individual domains domain, including the pore-forming S5 and S6 segments in the cytoplasmic region are compared, little change is and the voltage-sensor like (VSL) domain, undergoes an found within the Helical domain, Handle domain and overall small-degree counterclockwise rotation from the www.cell-research.com | Cell Research | SPRINGER NATURE Cryo-EM structures of RyR1 in multiple conformations closed to open state when viewed from the cytoplasmic On the other side, the transmembrane fragment close side (Figure 3B). to the cytoplasm and cytoplasmic extension of the S6 segment (designated the S6 segment hereafter) swing Cyt Cryo-EM structure of the open RyR1 outwards, resulting in the dilation of the intracellular gate The moderate resolution of the open RyR1 structure (Figure 4A, Supplementary information, Movie S2). disallowed side chain assignment. Nevertheless, the The distance between the Cα atoms of the constriction pronounced backbone shifts of S6 segments support the site residue Ile4937 in the diagonal protomers increases open conformation of the new structure as seen in the from 10.4 Å to 15.6 Å (Figure 4B). The calculated pore EM map (Figure 3B, Supplementary information, diameter of the constriction site in the high-resolution 2+ Figure S2B). When the structures of the open- and closed RyR1 was ~1.6 Å, blocking Ca passage. Now closed (C3)-RyR1 are overlaid relative to the channel that the diameter is expanded by ~5 Å, it would allow 2+ domain, the luminal halves including those of S5, S6, the permeation of single-file Ca even with hydration shell selectivity filter, and P loops, remain nearly unchanged. (Supplementary information, Movie S2). Figure 2 Conformational changes of the individual domains between C1 and C4 conformers. (A) Structural comparison be- tween C1 (gray) and C4 (violet) protomers. The two tetrameric structures are superimposed relative to the channel domain. The violet arrows indicate the conformational changes from C1 to C4. The same is applied to the other panels. (B) Structural shifts of the corona between C1 and C4 tetramers. The outskirt of the corona, composed of the Handle and Helical domains, moves towards the SR lumen from C1 to C4. Shown here is a side view. (C) The NTD undergoes a rocking motion with the central domains A and B upwards and the outer domain C downwards. For visual clarity, only two diagonal protomers are shown in side view. (D, E) The Central (D) and channel (E) domains remain nearly unchanged between C1 and C4 states. All structure figures were prepared with PyMol [48]. SPRINGER NATURE | Cell Research | Vol 26 No 9 | September 2016 Xiao-Chen Bai et al. To identify the deviation point of the S6 segments, the two structures are superimposed relative to the selectivity filter (SF) and the supporting helices (residues 4 836 to 4 935; Figure 4C). An ~15° bending of S6 helix occurs at a conserved Gly residue (Gly4934 in RyR1; Figure 4C). The structural observation supports a recent characterization that substitution of Gly4934 led to altered channel gating and ion conductance. Gly may provide the molecular basis for conformational flexibility [26]. Coupled conformational changes of the cytoplasmic O-ring of the channel domain As analyzed previously, the S6 segment, CTD, and Cyt the cytoplasmic segments of the VSL domain (designated the VSL domain) together constitute an O-ring (Figure Cyt 5A). When the channel domains in the structures of the open and C3 states are superimposed relative to the SF and the supporting helices, the elements in the cytoplas- Figure 3 The cryo-EM reconstruction of an open RyR1. (A) The mic O-ring appear to undergo concordant shift (Figures overall cryo-EM map of the open RyR1 at 5.7 Å resolution. Two 3B and 5A). Comparison of the individual domains perpendicular views are shown. (B) Comparison of the channel shows little intra-domain rearrangement within the VSL domain between the open and the C3 conformers. Shown on the right is the cytoplasmic view. The arrows indicate the confor- mational change from the C3 (blue) to the open (yellow) state. Figure 4 Dilation of the inner gate leads to channel opening. (A) The cytoplasmic gate of the S6 bundle undergoes a dilation that leads to pore opening, while the luminal segments have little change. (B) Dilation of the cytoplasmic gate. The indicated distances are measured between the Cα atoms of the gating residue Ile4937 on the S6 segments in the diagonal protomers. Please refer to Supplementary information, Movie S2 for the conformational changes of the channel domain. (C) Com- parison of the S6 segment relative to the luminal halves of the pore-forming segments (residues 4 836-4 935) shows that the structural deviation between the open (yellow) and closed (blue) states occurs at Gly4934. www.cell-research.com | Cell Research | SPRINGER NATURE Cryo-EM structures of RyR1 in multiple conformations or CTD domain (Figure 5B and 5C). In addition, there is and Ile4936 on S6 in the same protomer also remain no relative motion between the CTD and the C-terminal nearly the same in the open and C3 structures (Figure segment of S6, supporting our previous finding that the 5D, lower panel; Supplementary information, Movie presence of a zinc-finger motif at the joint of CTD and S3). S6 may rigidify the two structural moieties [23] (Figure The structural observations suggest that shifts of VSL 5C). and CTD may pull the S6 segments as well as the con- The extensive interactions between the S5 and S6 straining S4-5 segments outwards to open the intracellu- segments within one protomer and those between the lar gate. VSL and the pore-forming segments in the neighbouring protomer were analyzed in details in our previous report Coupled conformational changes between the cytoplas- [23]. We predicted that these extensive interactions may mic O-ring of the channel domain and the U-motif of the provide the molecular basis for coupled conformational Central domain changes. Indeed, comparison of the open and C3 struc- As shown previously, the cytoplasmic O-ring of the tures shows that these elements undergo coupled mo- channel domain accommodates the U-motif in the Cen- tion during pore opening (Figure 5D, Supplementary tral domain. The helical hairpin of the U-motif pierces information, Movie S3). For instance, the inter-helical through the O-ring, whereas the anti-parallel β-strands distances between S4 in one protomer and S5 in the ad- are located at the concave surface below the O-ring (Fig- jacent protomer in the C3 structure are similar to those ure 6A). The intricate interactions between the U-motif in the open structure (Figure 5D, upper panels). Simi- and the O-ring tie them into a stable unit. Indeed, the larly, the distances between the Cα atoms of Ile4826 on U-motif moves together with the O-ring during pore the S4-5 segment and Ile4931 on the S6 segment in the opening (Figure 6A). When the open and C3 structures neighbouring protomers and between Val4830 on S4-5 are superimposed relative to CTD, the U-motif can be Figure 5 Coupled conformational shifts of the segments within the channel domain during pore opening. (A) Structural com- parison of the channel domain in one protomer between the open and C3 structures relative to the luminal halves of the pore-forming segments (residues 4 836-4 935). The cytoplasmic “O-ring” composed of the cytoplasmic segments of S6 (S6 ), Cyt the VSL , and the CTD appears to undergo concordant shifts. (B) The VSL appears rigid during pore opening. There is no Cyt intra-domain rearrangement observed between the VSL domain in the open and closed structures. Therefore, the VSL do- main undergoes a rigid-body shift during pore opening. (C) There is no relative motion between CTD and the S6 segment Cyt during the pore dilation. (D) The concerted motions between the S5 and S6 segments in the same protomer, and between the S5 segment and the S4 and S4-5 segments in the neighbouring protomers. The distances between the C atoms of the indicated residues are presented in Å. For visual clarity, the specified protomer is coloured blue and yellow in the closed and open states, respectively, while the neighbouring protomer is coloured green in both states. The other two protomers that are not discussed are coloured gray. Please refer to Supplementary information, Movie S3 for the concordant shifts of the channel segments during pore dilation. SPRINGER NATURE | Cell Research | Vol 26 No 9 | September 2016 Xiao-Chen Bai et al. Figure 6 Coupled conformational changes between the channel domain and the Central domain. (A) Concordant confor- mational changes between the cytoplasmic “O-ring” of the channel domain and the U-motif of the Central domain. Shown here are the luminal and side views of the U-motif and the O-ring. The U-motif in the open RyR1 is coloured orange. Please refer to Supplementary information, Movie S4 for the concerted conformational changes. (B) Structural comparison of the O-ring and the U-motif between the C3 conformer and the open structure relative to CTD. There is little shift of the U-motif relative to CTD. (C) Comparison of the Central domain between the open and the C3 structures relative to the tetramer- ic channel domain (left panel) and relative to the individual Central domain (right panel). The tetrameric Central domain is shown in cytoplasmic view. The yellow and green arrows indicate the conformational transition from C3 to the open state. almost completely overlaid, indicating coupled motion may lead to the U-motif shift. between the CTD and the U-motif (Figure 6B). The Central domain undergoes a slight outward dis- The above analysis is based on the structure deviation placement from the closed to open conformation when between the open- and closed-RyR1 structures. In terms viewed from the SR lumen (Figure 6C). Within the Cen- of gating, the channel is pulled-open by signals applied tral domain, the U-motif and the nearby helix α20, as to the cytoplasmic region. Therefore, it is likely that the well as the extruding helix α4, slightly squeeze toward displacement of U-motif mobilizes the O-ring, leading the center of the concave side of the armadillo repeats, to the dilation of the S6 segments. We next analyzed the but there is little rearrangement of the armadillo repeats conformational changes of the cytoplasmic domains that (Figure 6C, right panel). The domainwise shift and the www.cell-research.com | Cell Research | SPRINGER NATURE Cryo-EM structures of RyR1 in multiple conformations intra-domain rearrangements of the Central domain like- surface of the Central domain and the amino terminal ly provide the pulling-force for the channel domain. We helices α1a and α1b in the Helical domain (Figure 7A). then examined the potential effect of the corona and pe- Similarly, the NTD also slightly revolves around the ripheral domains on the structural changes of the Central interface with the Central domain, but to the opposite domain. direction of the rotation of the Helical domain relative to the Central domain (Figure 7B). As the Handle domain Lateral rotation of the Central domain triggered by and the armadillo repeats of the NTD are consecutive, it structural shifts of the NTD, Handle and Helical domains is not surprising that the rotation of the Handle domain To understand the potential action of other cytoplas- is consistent with that of the NTD, i.e., centering around mic domains on the Central domain, we compared them the interface with the Central domain (Figure 7C). pairwise between the open and C3 structures (Figure 7A- In our previous analysis of the 3.8 Å structure of 7C, Supplementary information, Movie S4). When closed RyR1, we paid particular attention to the inter- the Central and Helical domains are compared relative action network among the super spiral assemblies in with the Central domain, the superspiral of the Helical the cytoplasmic region. Basically, all the armadillo re- domain appears to rotate around the interface between peats-containing domains contact each other (Figure 7D- the two domains involving the amino terminal concave 7F). They together constitute a network of pronounced Figure 7 The extensive interaction network among the cytoplasmic domains provide the molecular basis for long-range al- losteric gating. (A-C) The motions of the Helical domain, NTD, and Handle domain relative to the Central domain between the closed and open structures. The comparison is made relative to the Central domain (labeled with asterisk). In all panels, domains in C3 are blue, while those in the open structure are domain-coloured. (D) Extensive interfaces among the armadillo repeats-containing cytoplasmic domains, including the NTD, Helical, Handle, and Central domains. The NTD from the neigh- bouring protomer is also shown, coloured pale yellow and labeled NTD’. (E) The extensive internal interactions within one cytoplasmic superhelical assembly consisting of the armadillo repeats in NTD, the Handle domain, and the Central domain. Note that the Central domain also interacts with the NTD. (F) The extensive interaction network involving the Helical domain, the Handle domain, the Central domain in one protomer and the NTD in the adjacent protomer. Please refer to our previous publication [23] for detailed analysis of the cytoplasmic interaction network. SPRINGER NATURE | Cell Research | Vol 26 No 9 | September 2016 Xiao-Chen Bai et al. plasticity, which can transduce the conformational chang- structures and between the closed and open structures es initiated to any point of the helical surface. The col- are compared (Supplementary information, Movies lective motions of the Helical domain, the NTD, and the S1 and S4). It is evident that the corona, peripheral do- Handle domain may lead to the observed compression of mains, and NTD of the cytoplasmic region undergo ver- the Central domain toward its concave side (Figure 6C, tical motions during the conformational changes between right panel). In addition to the internal rearrangement of the distinct closed states (Supplementary information, the Central domain, an overall concerted lateral rotation Movie S1). In contrast, despite the overall similarity of the Central and the other cytoplasmic domains occur between the open and C3 structures, the lateral rotation between the open and closed states when viewed from of the cytoplasmic domains is evident (Supplementary the side (Supplementary information, Movie S4). information, Movie S4). As illustrated above, the pore opening requires dilation Discussion of the S6 helical bundle at the intracellular gate (Figure 4). The shift of S6 is triggered by the motion of VSL and Cyt In this study, we report the structures of RyR1 in three CTD (Figure 5), which is induced by the displacement of closed states and an open state. It is particularly interest- the U-motif in the Central domain (Figure 6). The shift ing when the conformational changes among the closed of the U-motif results from both intra-domain rearrange- Figure 8 Speculative mechanism of the excitation-contraction coupling. (A) Conformational changes to any cytoplasmic domain may be propagated to the Central domain along the interaction network described in Figure 7. Shown here are side views of tetrameric RyR1. Inset: an example of a speculative route (black arrows) for the propagation of conformational changes that can be triggered by motion of the SPRY3 domain. (B) Speculative model of the complex between RyR1 and the Ca 1.1 complex. Structural determination of both RyR1 and the Ca 1.1 complex provides the foundation for elucidating the v v molecular mechanism of RyR1 opening induced by depolarization of the plasma membrane. Structures of the Ca 1.1 com- plex (PDB code: 3JBR) [34] and RyR1 were manually docked in COOT [43]. www.cell-research.com | Cell Research | SPRINGER NATURE Cryo-EM structures of RyR1 in multiple conformations The fractions containing RyR1-FKBP12 complex were pooled for ment and overall lateral rotation of the Central domain. EM analysis. Before loading to grids for cryo sample preparation, In essence, the pore opening requires mobilization of the the complex was incubated with 50 µM CaCl and 10 µM PCB95 Central domain, which thereby serves as the transducer for 30 min on ice. The sample that gave rise to the open struc- of the long-range conformational changes. ture was purified with 0.5% CHAPS (w/v; Amresco) instead of Under physiological condition, RyR1 is activated Tween-20. The other procedures were the same. through direct physical contacts with the Ca 1.1 complex as well as the surrounding RyR1 tetramers in the crystal- Cryo-EM image acquisition Aliquots of 3 µl purified RYR1 at a concentration of ~30 nM line-like assembly [27-32]. It was shown that multiple ar- were placed on glow-discharged holey carbon grids (Quantifoil eas of the RyR1 cytoplasmic region, such as the SPRY3 Cu, R2/2), on which a home-made continuous carbon film (esti - domain, are involved in the coupling with Cav1.1 com- mated to be ~30 Å thick) had previously been deposited. Grids plex (Figure 8A) [33]. Note that the SPRY3 domain is in were blotted for 2 s and flash-frozen in liquid ethane using an FEI direct contact with NTD. Potential shifts of the SPRY3 Vitrobot II. Grids were transferred to an FEI Tecnai Polara electron domain may be translated to the conformational changes microscope that was operating at 300 kV. Images were recorded of NTD, and subsequently the Handle domain and the manually using a prototype FEI Falcon-III detector at a calibrated Central domain. We speculate that the shift of SPRY3 magnification of 104 478, yielding a pixel size of 1.34 Å. A dose rate of 20 electrons/Å /s, and an exposure time of 2 s were used on may involve a displacement to trigger the lateral rotation the Falcon. of the cytoplasmic domains of RyR1 (Figure 8A, inset). Although the elements in the Ca 1.1 complex that bind Image processing to RyR1 remain to be elucidated, the recent structural de- Similar image processing procedures were employed as report- termination of the Ca 1.1 complex laid out the foundation ed [23]. We used MOTIONCORR [35] for whole-frame motion for investigation of the gating mechanism of RyR1 (Fig- correction, CTFFIND3 [36] for estimation of the contrast transfer ure 8B) [34]. Structures of the complex between Ca 1.1 function parameters, and RELION-1.4 [37] for all subsequent steps. References for template-based particle picking [38] were and RyR1 as well as the structures of Ca 1.1 in multiple obtained from 2D class averages that were calculated from a man- states would offer the answer to address the fundamental ually picked subset of the micrographs. A 20 Å low-pass filter was problem of how depolarization of the plasma membrane applied to these templates to limit model bias. To discard false pos- would induce the pore opening of RyR1, which resides in itives from the picking, we used initial runs of 2D and 3D classifi- the SR membrane. We speculate that the conformation- cation to remove bad particles from the data. The selected particles al changes of the voltage-sensing domains of Ca 1.1α1 v were then submitted to 3D auto-refinement, particle-based motion upon depolarization would induce shifts of the β-subunit correction and radiation-damage weighting [38]. The resulting “polished particles” were used for masked classification only on and other cytoplasmic segments of Ca 1.1, which may pore region with subtraction of the residual signal [35], and the trigger the motion of the adjoining RyR1 cytoplasmic do- original particle images from the resulting classes were submitted mains exemplified by the SPRY3 domain. The structural to a second round of 3D auto-refinement. All 3D classifications shifts at the periphery of the RyR1 cytoplasmic region and 3D refinements were started from a 40 Å low-pass filtered ver - are propagated along the superhelical assemblies of the sion of the high-resolution consensus structure. Fourier Shell Co- cytoplasmic domains to the Central domain, eventual- efficient (FSC) curves were corrected for the effects of a soft mask ly leading to the opening of the intracellular gate. The on the FSC curve using high-resolution noise substitution [39]. speculative mechanism awaits experimental evidence. Reported resolutions are based on gold-standard refinement pro- cedures and the corresponding FSC = 0.143 criterion [40]. Prior to In addition, high resolution structures of RyR channels visualization, all density maps were corrected for the modulation in various states are required to reveal the modulation of 2+ transfer function of the detector, and then sharpened by applying a the channel activity by multiple signals such as Ca and negative B-factor that was estimated using automated procedures PCB95. [41]. 2+ For the sample purified in TWEEN-20/PCB95/Ca , 334K Materials and Methods particles were selected after initial 2D and 3D classification. Subsequent 3D auto-refinement and particle polishing yielded a map with relatively fuzzy densities in the cytoplasmic region. 3D Protein purification The RyR1-FKBP12 complex that was captured in multiple classification into five classes with small angular sampling yielded closed conformations was purified following similar protocol as three classes with better density of the cytoplasmic region. Rel- before with slight modifications [23]. The buffer for the last step atively poor reconstructed density was observed in the other two size-exclusion chromatography (Superdex-200, 10/30, GE Health- classes. Separate 3D auto-refinements of the corresponding parti - care) purification was changed to 20 mM MOPS-Na, pH 7.4, 250 cles in the original data set for the three best classes gave rise to mM NaCl, 2 mM DTT, 0.015% Tween-20 (w/v; Sigma-Aldrich) reconstructions to 3.8-4.2 Å resolution (also see Supplementary and protease inhibitor cocktail including 2 mM PMSF, 2.6 µg/ml information, Figure S1 and Table S1). 2+ aprotinin, 1.4 µg/ml pepstatin, and 10 µg/ml leupeptin (Amresco). For the sample purified in CHAPS/PCB95/Ca , initial classifi- SPRINGER NATURE | Cell Research | Vol 26 No 9 | September 2016 Xiao-Chen Bai et al. cation selected 46K particles. After particle polishing, application tion in calcium release. Cold Spring Harb Perspect Biol 2010; of the masked classification procedure on the pore region with 2:a003996. residual signal subtraction into three classes identified a single 5 Takeshima H, Nishimura S, Matsumoto T, et al. Primary class with good density and open conformation. After 3D auto-re- structure and expression from complementary DNA of skele- finement, the corresponding 30K particles gave a map with a reso- tal muscle ryanodine receptor. Nature 1989; 339:439-445. lution of 5.7 Å. 6 Rossi D, Sorrentino V. Molecular genetics of ryanodine recep- tors Ca2+-release channels. Cell Calcium 2002; 32:307-319. 7 Otsu K, Willard HF, Khanna VK, Zorzato F, Green NM, Ma- Model building and refinement The initial model (PDB: 3J8H) was docked into each map by cLennan DH. Molecular cloning of cDNA encoding the Ca2+ using DireX [42]. The resulting models were manually adjusted release channel (ryanodine receptor) of rabbit cardiac muscle in COOT [43] to further improve the fitting of secondary struc - sarcoplasmic reticulum. J Biol Chem 1990; 265:13472-13483. tures and side chains. Subsequently, all models were refined using 8 Nakai J, Imagawa T, Hakamat Y, Shigekawa M, Takeshima REFMAC [44] with secondary structure restraints generated by H, Numa S. Primary structure and functional expression from ProSMART [45]. To prevent overfitting, the optimal weight for cDNA of the cardiac ryanodine receptor/calcium release chan- nel. FEBS Lett 1990; 271:169-177. refinement in REFMAC were determined by cross-validation [46]. 9 Hakamata Y, Nakai J, Takeshima H, Imoto K. Primary struc- ture and distribution of a novel ryanodine receptor/calcium Accession codes The atomic coordinates of the C1, C3, C4, and open-RyR1 release channel from rabbit brain. FEBS Lett 1992; 312:229- structures have been deposited in the Protein Data Bank with the 235. accession codes 5GKY, 5GKZ, 5GL0, and 5GL1, respectively. The 10 Radermacher M, Wagenknecht T, Grassucci R, et al. Cryo- cryo-EM maps have been deposited to EMDB with the following EM of the native structure of the calcium release channel/ accession codes: EMD-9518 (C1), EMD-9519 (C3), EMD-9520 ryanodine receptor from sarcoplasmic reticulum. 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Structure of the voltage-gated calci- transmit the Contribution as long as it attributed back um channel Cav1.1 complex. Science 2015; 350:aad2395. to the author. Readers are permitted to alter, transform 35 Bai XC, Rajendra E, Yang G, Shi Y, Scheres SH. Sampling or build upon the Contribution as long as the resulting work is then the conformational space of the catalytic subunit of human distributed under this is a similar license. Readers are not permitted gamma-secretase. eLife 2015; 4:e11182. to use the Contribution for commercial purposes. Please read the full 36 Mindell JA, Grigorieff N. Accurate determination of local de- license for further details at - http://creativecommons.org/ licenses/by- focus and specimen tilt in electron microscopy. J Struct Biol nc-sa/4.0/ 2003; 142:334-347. 37 Scheres SH. RELION: implementation of a Bayesian ap- © The Author(s) 2016 proach to cryo-EM structure determination. 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Published: Jul 29, 2016

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