Modulation of cardiac ryanodine receptor 2 by calmodulin

Abstract

The high-conductance intracellular calcium (Ca2+) channel RyR2 is essential for the coupling of excitation and contraction in cardiac muscle. Among various modulators, calmodulin (CaM) regulates RyR2 in a Ca2+-dependent manner. Here we reveal the regulatory mechanism by which porcine RyR2 is modulated by human CaM through the structural determination of RyR2 under eight conditions. Apo-CaM and Ca2+-CaM bind to distinct but overlapping sites in an elongated cleft formed by the handle, helical and central domains. The shift in CaM-binding sites on RyR2 is controlled by Ca2+ binding to CaM, rather than to RyR2. Ca2+-CaM induces rotations and intradomain shifts of individual central domains, resulting in pore closure of the PCB95 and Ca2+-activated channel. By contrast, the pore of the ATP, caffeine and Ca2+-activated channel remains open in the presence of Ca2+-CaM, which suggests that Ca2+-CaM is one of the many competing modulators of RyR2 gating.

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Fig. 1: Cryo-EM structures of the RyR2–CaM complexes.
Fig. 2: Interfaces between CaM and RyR2.
Fig. 3: Molecular basis for the shift of binding site for CaM after Ca2+ loading.
Fig. 4: Schematic of RyR2 modulation by CaM.

Data availability

Atomic coordinates and electron microscopy density maps of the following structures have been deposited in the PDB (http://www.rcsb.org) and the Electron Microscopy Data Bank (EMDB https://www.ebi.ac.uk/pdbe/emdb/). FKBP12.6/apo-CaM (PDB, 6JI8; EMDB, EMD-9833), FKBP12.6/ATP/caffeine/low-[Ca2+]/CaM-M (PDB, 6JII; EMDB, EMD-9834), FKBP12.6/ATP/caffeine/low-[Ca2+] (PDB, 6JI0; EMDB, EMD-9831), FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM (PDB, 6JIU; EMDB, EMD-9836), CHAPS- and DOPC-treated FKBP12.6/ATP/caffeine/low-[Ca2+] (PDB, 6JRR; EMDB, EMD-9879), CHAPS- and DOPC-treated FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM (PDB, 6JRS; EMDB, EMD-9880), FKBP12.6/ATP/caffeine/high-[Ca2+]/Ca2+-CaM (PDB, 6JIY; EMDB, EMD-9837) and PCB95/low-[Ca2+]/Ca2+-CaM (PDB, 6JV2; EMDB: EMD-9889) complexes. Source Data for Fig. 2e, f and Extended Data Figs. 1c, 6f, 7d–h are available in the online version of the paper. All other data are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank X. Li for technical support for electron microscopy image acquisition; the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) for providing the cryo-EM facility support; the computational facility support on the cluster of Bio-Computing Platform (Tsinghua University Branch of China National Center for Protein Sciences Beijing) and the ‘Explorer 100’ cluster system of Tsinghua National Laboratory for Information Science and Technology; M. T. Overgaard for providing the CaM protein for single channel studies. This work was funded by the National Key R&D Program (2016YFA0500402) and the National Key Basic Research (973) Program (2015CB910101) from Ministry of Science and Technology of China and the National Natural Science Foundation of China (projects 31621092, 31630017 and 81861138009). N.Y. is supported by the Shirley M. Tilghman endowed professorship from Princeton University. This work was also supported by research grants from the Heart and Stroke Foundation of Canada, the Canadian Institutes of Health Research and the Heart and Stroke Foundation Chair in Cardiovascular Research (S.R.W.C.).

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Contributions

D.G. and N.Y. conceived the project. D.G., X.C. and G.Z. prepared the electron microscopy samples. D.G., X.C., G.H. and J.L. conducted the cryo-EM analysis. J.W., L.Z. and R.W. performed the functional experiments. All authors contributed to data analysis. D.G., S.R.W.C. and N.Y. wrote the manuscript.

Corresponding authors

Correspondence to Deshun Gong or S. R. Wayne Chen or Nieng Yan.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Protein purification and structural determination.

a, Schematic of vector construction for recombinant expression of CaM without the N-terminal Met. b, N-terminal sequencing confirmed removal of the initial Met. c, SEC purification of the affinity-purified complex of pRyR2–FKBP12.6 (containing GST–FKBP12.6). The experiment was repeated five times independently with similar results. Peak fractions were resolved by SDS–PAGE and visualized by Coomassie blue staining (Supplementary Fig. 1). UV, ultraviolet. MWM, molecular weight marker. d, The channel is open in the presence of ATP, caffeine and Ca2+ under both digitonin and CHAPS-and-DOPC (indicated by an asterisk) conditions. The pore of CHAPS- and DOPC-treated FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM remains open after Ca2+-CaM loading. The ion-conduction path, calculated by HOLE, is illustrated by dots in each structure. A, ATP; C, caffeine; Ca2+-CaM, Ca2+-bound CaM; CaM-M, a Ca2+-binding-deficient CaM mutant that mimics apo-CaM; F, FKBP12.6; L-Ca2+, low Ca2+ concentration. e, Overall electron microscopy map of the FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM complex. Inset, the cytoplasmic view of the channel gate. Because the side chains of the Gln4864-gating residues are not well-resolved, the distance between the Cα atoms of Gln4864 gating in the diagonal protomers is shown in the dashed circle. The density corresponding to CaM was generated from the map that was low-pass-filtered to 5.6 Å with a contour level of 0.015; the other regions were from the 4.2 Å map with a contour level of 0.023. f, Overall electron microscopy map of the CHAPS- and DOPC-treated FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM complex. The density corresponding to CaM was generated from the map that was low-pass-filtered to 5.6 Å with a contour level of 0.013; the other regions were from the 3.7 Å map with a contour level of 0.021. g, Although the concentrations of Ca2+-CaM are the same in these three conditions, only the N-lobe can be resolved in the FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM and CHAPS- and DOPC-treated FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM RyR2 structures. The reason may be that the HD2 in these two structures presents a steric hindrance for C-lobe binding. P, PCB95. h, The corresponding pore radii of the three structures are plotted. i, j, Gold-standard Fourier shell correlation curves for electron microscopy maps of the eight datasets. H-Ca2+, high Ca2+ concentration.

Extended Data Fig. 2 Flow chart for cryo-EM data processing.

See Methods for details. a, Data processing of the FKBP12.6/apoCaM, FKBP12.6/ATP/caffeine/low-[Ca2+]/CaM-M, CHAPS- and DOPC-treated FKBP12.6/ATP/caffeine/low-[Ca2+], and PCB95/low-[Ca2+]/Ca2+-CaM datasets. b, Data processing of the FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM, FKBP12.6/ATP/caffeine/high-[Ca2+]/Ca2+-CaM, FKBP12.6/ATP/caffeine/low-[Ca2+], and CHAPS- and DOPC-treated FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM datasets.

Extended Data Fig. 3 Local resolution maps of the eight reconstructions.

a, c, e, g, i, k, l, n, The local resolution maps estimated with RELION 2.0. All electron microscopy maps were generated in Chimera and contoured at levels of 0.027 (a), 0.022 (c), 0.023 (e), 0.021 (g), 0.02 (i), 0.021 (k), 0.015 (l) and 0.021 (n). b, Electron microscopy map of apo-CaM from the reconstruction shown in a. d, Electron microscopy map of CaM-M. f, h, The electron microscopy densities of Ca2+-CaM were generated from the maps of FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM and CHAPS- and DOPC-treated FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM that were low-pass-filtered to 5.6 Å with contour levels of 0.015 (f) and 0.013 (h), respectively. j, m, Electron microscopy densities of Ca2+-CaM in FKBP12.6/ATP/caffeine/high-[Ca2+]/Ca2+-CaM (j) and PCB95/low-[Ca2+]/Ca2+-CaM (m). The densities of both lobes were resolved in the map, although the N-lobe was resolved better than the C-lobe.

Extended Data Fig. 4 Representative local electron microscopy maps for FKBP12.6/apo-CaM and densities of the binding sites for Ca2+, ATP and caffeine in FKBP12.6/ATP/caffeine/high-[Ca2+]/Ca2+-CaM.

ai, The electron microscopy maps for the representative segments of RyR2. All of the maps were contoured at 5.5σ. j, The binding sites for Ca2+, ATP and caffeine in RyR2. The blue, dotted circle indicates the O-ring that is formed by the C-terminal subdomain (CTD), cytoplasmic subdomain in the voltage-sensor-like domain (VSC) and the cytoplasmic portion of S6 (S6C). Ca2+ is located in the cleft that is formed by the central domain and C-terminal subdomain. ATP is located in a pocket formed by the U-motif, C-terminal subdomain and S6C. Caffeine is located at the interface formed by the U-motif, helix α4, C-terminal subdomain and voltage-sensor-like domain. The red letter indicates a disease-causing variant. km, The local densities of the Ca2+-, ATP- and caffeine-binding sites. The electron microscopy maps of the FKBP12.6/ATP/caffeine/high-[Ca2+]/Ca2+-CaM (+ATP, caffeine and Ca2+) and FKBP12.6/apo-CaM (−ATP, caffeine and Ca2+) RyR2 structures are shown. All of the electron microscopy maps were contoured at a level of 0.029.

Extended Data Fig. 5 Binding interfaces between CaM and RyR2.

a, Three interfaces are formed between the N-lobe of apo-CaM and RyR2. HD1 serves as the major binding site for the N-lobe. The Cα atom of Lys2558 is shown as a sphere. b, c, Two interfaces are formed between the C-lobe of apo-CaM and RyR2. Helix α−1 is the major binding site of the C-lobe. d, e, The interfaces between the N-lobe of Ca2+-CaM and RyR2. f, The interface between the C-lobe of Ca2+-CaM and RyR2. g, Local densities of the probable interacting residues, Tyr2157, Tyr2203, Arg2206 and Lys2154 in RyR2. The electron microscopy map was contoured at 5.5σ. h, Density of helix α−1 in the FKBP12.6/apo-CaM RyR2 structure. The sequence can be reliably assigned based on the indicated bulky residues. i, Density of helix α−1 in the FKBP12.6/ATP/caffeine/high-[Ca2+]/Ca2+-CaM RyR2 structure. The C-terminal half of helix α−1 is reliably assigned, a few bulky residues facilitate the sequence alignment. As both the N-terminal half of helix α−1 and C-lobe of Ca2+-CaM had a lower resolution, the density shown here may belong to Trp3588. The electron microscopy maps in h and i were contoured at levels of 0.027 and 0.016, respectively.

Extended Data Fig. 6 Effect of RyR2 mutations on CaM regulation of single RyR2 channels.

ae, Single-channel activities were recorded in a symmetrical recording solution containing 250 mM KCl and 25 mM HEPES, pH 7.4. Representative current traces of single RyR2(WT) (n = 9), RyR2(Y2156A) (n = 8), RyR2(V3599A) (n = 7), RyR2(W3587A) (n = 8) and RyR2(L3590A) (n = 9) channels are shown. The Ca2+ concentration on the cytoplasmic and luminal face of the channel was 440 nM and around 45 nM, respectively. Open probability (Po), mean open time (To) and mean closed time (Tc) of the same channel before and after addition of CaM(WT) (1 μM) are depicted. Baselines are indicated by short bars on the right. f, Percentages of inhibition of channel open probability by CaM. Data are mean ± s.e.m. from single RyR2(WT) (n = 9), RyR2(Y2156A) (n = 8), RyR2(V3599A) (n = 7), RyR2(W3587A) (n = 8) and RyR2(L3590A) (n = 9) channels and analysed by one-way ANOVA with a Dunnett’s post hoc test (versus RyR2(WT)) and adjusted P values are indicated on the graph. Source Data

Extended Data Fig. 7 Effect of RyR2 and CaM mutations on the termination of Ca2+ release in HEK293 cells.

HEK293 cell lines that express RyR2(WT) and RyR2 mutants were co-transfected with the FRET-based endoplasmic-reticulum luminal Ca2+-sensing protein D1ER and with no CaM (control), CaM(WT) or the Ca2+-binding-deficient CaM mutant, CaM-M. ac, Representative single-cell luminal Ca2+ recordings of RyR2(WT) cells transfected with no CaM (a; n = 155 cells), RyR2(WT) cells transfected with CaM(WT) (b; n = 178 cells) and RyR2-WT cells transfected with CaM-M (c; n = 177 cells) are shown. FSOICR indicates the FRET level at which SOICR occurs and Ftermi represents the FRET level at which SOICR terminates. The signal Fmax is defined as the FRET level after tetracaine treatment. The minimum FRET signal maximum FRET Fmin is defined as the FRET level after caffeine treatment. d, The activation threshold was determined as shown in a. e, The store capacity was calculated by subtracting Fmin from Fmax. d, e, Data are mean ± s.e.m. with the number of independent experiments for each condition shown on the graph and analysed by one-way ANOVA with a Dunnett’s post hoc test (versus RyR2(WT) control) and adjusted P values are indicated. fh, RyR2(WT) cells were co-transfected with the FRET-based endoplasmic-reticulum luminal Ca2+-sensing protein D1ER and CaM(WT) or CaM mutants (CaM-M, CaM(E15A), CaM(F66A), CaM(L70A), CaM(M110A), CaM(F142A), CaM(F20A), CaM(F69A), CaM(F93A), CaM(L106A) and CaM(M146A)). CaM mutations close to a specific CaM–RyR2 interface are grouped and are indicated. The termination threshold (f), activation threshold (g) and store capacity (h) were determined as described in ae above. fh, Data are mean ± s.e.m. with the number of independent experiments for each condition shown on the graph and analysed by one-way ANOVA with a Dunnett’s post hoc test (versus CaM(WT)) and adjusted P values are indicated. Source Data

Extended Data Fig. 8 Evaluation of the conformations of N- and C-lobes of CaM in the FKBP12.6/ATP/caffeine/high-[Ca2+]/Ca2+-CaM structures.

a, The electron microscopy map (low-pass-filtered to 4.8 Å resolution at a contour level of 0.015) of FKBP12.6/ATP/caffeine/high-[Ca2+]/Ca2+-CaM. Red and blue boxes indicate the N- and C-lobes, respectively. b, Docking of the three reported conformations of the N-lobe of CaM into our electron microscopy map suggests an open conformation of the N-lobe in FKBP12.6/ATP/caffeine/high-[Ca2+]/Ca2+-CaM. CC indicates the cross-correlation coefficient. c, An important distinction between C-lobes in the open and semi-open or closed states is the enlarged angle between helices C1 and C4. d, Docking analysis supports the open conformation of the C-lobe in FKBP12.6/ATP/caffeine/high-[Ca2+]/Ca2+-CaM.

Extended Data Fig. 9 Inhibitory mechanism of RyR2 by Ca2+-CaM.

a, Both caffeine and ATP are located at the interfaces between the U-motif and O-ring, locking these elements into a stable unit. b, There is almost no intradomain rearrangement of the individual central domain between the structures of FKBP12.6/ATP/caffeine/low-[Ca2+] and FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM. c, Ca2+-CaM induces anticlockwise rotation of the overall central domain in FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM when viewed from the cytoplasmic side. The overall tetrameric FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM and FKBP12.6/ATP/caffeine/low-[Ca2+] RyR2 structures are superimposed relative to C-terminal subdomain of the channel domain. Red arrows indicate the conformational changes upon Ca2+-CaM binding. d, Ca2+-CaM induces intradomain shifts of the individual central domain in the PCB95 and Ca2+-activated channel. e, The PCB95 and Ca2+-activated  channel closes after Ca2+-CaM loading.

Extended Data Fig. 10 Conformational changes induced by Ca2+-CaM and mapping of previously identified CaM-binding sequences and disease-associated point mutations onto the structures of RyR2–CaM complexes.

a, Compared to FKBP12.6/ATP/caffeine/low-[Ca2+], the four central domains in the FKBP12.6/ATP/caffeine/high-[Ca2+]/Ca2+-CaM RyR2 structure undergo an anticlockwise rotation. The overall tetrameric FKBP12.6/ATP/caffeine/low-[Ca2+] and FKBP12.6/ATP/caffeine/high-[Ca2+]/Ca2+-CaM RyR2 structures are superimposed relative to the C-terminal subdomain of the channel domain. b, Compared to the CHAPS- and DOPC-treated FKBP12.6/ATP/caffeine/low-[Ca2+] RyR2 structure, almost no conformational change was induced by Ca2+-CaM in the CHAPS- and DOPC-treated FKBP12.6/ATP/caffeine/low-[Ca2+]/Ca2+-CaM RyR2 structure. c, Compared to the PCB95/low-[Ca2+] RyR2 structure, the overall central domain in the PCB95/low-[Ca2+]/Ca2+-CaM RyR2 structure undergoes an anticlockwise rotation. The overall tetrameric PCB95/low-[Ca2+] and PCB95/low-[Ca2+]/Ca2+-CaM RyR2 structures are superimposed relative to the C-terminal subdomain of the channel domain. d, Structural mapping of previously reported CaM-binding sequences. Orange, the overlapping binding sequences of apo-CaM and Ca2+-CaM; cyan, the binding sequence of Ca2+-CaM; yellow, the binding sequence of apo-CaM; blue, segments that are not involved in binding in our structures; red, sequences that are invisible in the structures. The residue numbers in brackets that are labelled grey indicate that the sequences are invisible in the structure. e, The primary apo-CaM binding sequences in RyR2 are the same in RyR1. Red residues highlight the key contact residues. f, Mapping of the disease-associated point mutations onto the structure of the RyR2–apo-CaM complex. The mutations in HD1 and apo-CaM are coloured blue and red, respectively. g, Mapping of the CaM disease-associated point mutations onto the structure of the RyR2–Ca2+-CaM complex.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

41586_2019_1377_MOESM3_ESM.mp4

Video 1 Rotation of the Central domains induced by Ca2+-CaM. Structures of the tetrameric F/A/C/L-Ca2+ and F/A/C/L-Ca2+/Ca2+-CaM are superimposed relative to the CTD of the Channel domain. Shown here are the four Central domains in the cytoplasmic view. The morph was generated in PyMol.

Supplementary Information

Supplementary Figure 1 and Supplementary Table 1.

Reporting Summary

Video 1 Rotation of the Central domains induced by Ca2+-CaM. Structures of the tetrameric F/A/C/L-Ca2+ and F/A/C/L-Ca2+/Ca2+-CaM are superimposed relative to the CTD of the Channel domain. Shown here are the four Central domains in the cytoplasmic view. The morph was generated in PyMol.

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Gong, D., Chi, X., Wei, J. et al. Modulation of cardiac ryanodine receptor 2 by calmodulin. Nature 572, 347–351 (2019). https://doi.org/10.1038/s41586-019-1377-y

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