Structural basis for ryanodine receptor type 2 leak in heart failure and arrhythmogenic disorders

Figures

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  Fig. 1. Cryo-EM reconstructions of human RyR2-S2808D: Rycal ARM210 stabilizes the closed state while calstabin-2 depletion induces the primed state. a Overlapped models of closed RyR2 (PDB:7UA5, gray) and open RyR2 (PDB:7UA9, yellow). The arrows show that the cytoplasmic shell of the channel shifts downward-outward when going from the closed to the open state. To facilitate visualization only the front protomer is shown in colors, while the other three protomers are shown as gray transparent volumes. The sarcoplasmic reticulum membranes are shown as black discs. b Overlapped models of closed RyR2 (PDB:7UA5, gray) and subprimed RyR2-S2808D (PDB:8UQ2, magenta). The arrows show that the cytoplasmic shell of the channel shifts downward-outward when going from the closed to the subprimed state. c RMSD normalized projection of WT RyR2 and RyR2-S2808D channels with different treatments. Values close to 0 or 1 indicate the conformation of the cytoplasmic shell is more similar to the closed WT RyR2 (PDB:7UA5), or the open WT RyR2 (PDB:7UA9), respectively. Negative values indicate the conformation of the cytoplasmic shell is more upward-inward than the closed state. d Overlapped models of…

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  Fig. 2. Cryo-EM reconstructions of human RyR2 variants show that the primed state is reversed by treatment with the Rycal ARM210. RMSD-normalized projections of the primed PKA-phosphorylated CPVT and HF variants in the absence and presence of ARM210. Values close to 0 or 1 indicate the conformation of the cytoplasmic shell is more similar to the closed WT RyR2, or the open WT RyR2, respectively. Negative values indicate the conformation of the cytoplasmic shell is more upward-inward than the closed state.

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  Fig. 3. R420Q/W mutations induce NTD-B domain rotation. a–c Aligned models focused on the NTD-B domain of closed PKA-phosphorylated RyR2 (PDB:7U9Q, gray), open PKA-phosphorylated RyR2 (PDB:7U9R, yellow, a), primed PKA-phosphorylated RyR2-R420Q (PDB:8UXC, magenta, b), and primed PKA-phosphorylated RyR2-R420W (PDB:8UXF, magenta, c). Models were aligned at the NSol domain. Conformational changes are indicated with arrows. d–f The same models with overlapping cryo-EM maps around residue 420 of closed PKA-phosphorylated RyR2 (d), primed PKA-phosphorylated RyR2-R420Q (e), and primed PKA-phosphorylated RyR2-R420W (f). R420 interacts with R298 and V300, generating a network of stabilizing interactions. The presence of smaller residues in the mutants leads to the collapse of the 298-303 loop. Conformational changes of the 298-303 loop are indicated using arrows, and distances are provided.

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  Fig. 4. Reduced calstabin-2 binding to the primed state of CPVT variants. a Binding curves of calstabin-2 to WT RyR2. Concentration of calstabin-2 is 5, 10, 50, 100, 500, and 1000 nM (from black to magenta), and RyR2 is 5 nM. Reactions were performed as duplicates. Data are presented as mean values and error bars as SD. All points were simultaneously fitted using the equation Association Kinetics - Two or more concentrations of ligand in GraphPad. Fitting curves are shown in gray, R2 = 0.9895. b Table showing the values of kon, koff, and Kd derived from the association kinetics curves for the different variants of RyR2. c Table showing the correlation between the primed state conformation (“RMSD”), calstabin-2 koff, and onset age for the CPVT mutants tested (y.o.: years old)53. “RMSD” stands for the value of the normalized RMSD difference analysis of each structure. *The age of onset for WT RyR2 was considered as the average age when exercise-induced ventricular arrhythmias begin to be significant in the asymptomatic population86,87. d, e Correlation between Kd and age of onset (d) and between koff and age of onset (e) for the variants tested. Reactions were performed as duplicates. Data…

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  Fig. 5. RyR2-R420W increased sensitivity to Ca2+ is partially reversed by CaM binding. a Cryo-EM particle population percentage in the samples of PKA-phosphorylated RyR2-R420W + Ca2+ in the absence and presence of CaM. b Overlapping models of primed PKA-phosphorylated RyR2-R420W (PDB:8UXF, gray) and primed PKA-phosphorylated RyR2-R420W + Ca2+ (PDB:8UXH, magenta). The arrows show that the cytoplasmic shell of the channel shifts downward-outward in the presence of Ca2+ inducing a more pronounced primed state. c Overlapping models of primed PKA-phosphorylated RyR2-R420W + Ca2+ (PDB:8UXH, magenta) and primed PKA-phosphorylated RyR2-R420W + Ca2+ + CaM (PDB:8UXL, cyan). The arrows show that the cytoplasmic shell of the channel shifts upward-inward in the presence of CaM, partially reversing the changes introduced by Ca2+. d RMSD normalized projection of RyR2-R420W + Ca2+ different states. Values close to 0 or 1 indicate the conformation of the cytoplasmic shell is more similar to the closed WT RyR2, or the open WT RyR2, respectively. e–g Cryo-EM maps of primed PKA-phosphorylated RyR2-R420W (e, gray), primed PKA-phosphorylated RyR2-R420W + Ca2+ (f, magenta), and primed PKA-phosphorylated…

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  Fig. 6. Regulation of RyR gating by the cytoplasmic shell. a Schematic representation of the conformational space of the cytoplasmic shell. b, c Schematic view of the dynamic energy landscape of the flexion component (b) and pore state component (c) of the conformational space of the cytoplasmic shell. In the flexion component, all conformations are energetically equal in the basal state (black). Binding of the ligands creates an alternative landscape that penalizes (green) or favors (magenta) the most downward-outward conformations. RyR2 can jump between two landscapes by ligand binding/unbinding or post-translational modifications (phosphorylation/dephosphorylation). In the pore state component, in the absence of activating Ca2+, the global minimum is the closed state with upward-inward conformations of the cytoplasmic shell. In the presence of activating Ca2+, the global minimum is the open state with downward-outward conformations of the cytoplasmic shell. d, e Schematic view of the dynamic energy landscape of the conformational space of the cytoplasmic shell (sum of both components), which explains the regulatory control of the cytoplasmic shell on the population distribution of…

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  Fig. 7. Proposed structural mechanisms of leaky RyR2 and its involvement in disease. a, b Schematic representation of the normal function of RyR2 (a) and the pathological disease-related disfunction (b). In the case of disease variants, the resting state is in a subprimed/primed state, which correlates with the higher calstabin-2 koff and Ca2+ leak during diastole under stress conditions. The orange object labeled “Cal” stands for calstabin. Figure 7a,b adapted from Marco C. Miotto et al., Structural analyzes of human ryanodine receptor type 2 channels reveal the mechanisms for sudden cardiac death and treatment. Sci. Adv. 8,eabo1272(2022). 10.1126/sciadv.abo1272 under a CC BY license: https://creativecommons.org/licenses/by/4.0/.10 c Vicious cycle involved in heart failure progression and arrhythmias. Figure 7c was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en.