Dynamic-Structure Redesign of Calmodulin Reveals Mechanistic Constraints on Ryr2 Regulation

Figures

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  Figure 1.. Mechanism of CaM-Mediated RyR2 Regulation and Dynamic-Structure Redesign. (A) Dynamic model of CaM–RyR2 regulation across the cardiac cycle. Top: Modeled conformational transitions of the CaM–RyR2 complex depict rearrangements of the N-domain (blue) and C-domain (magenta) during systolic activation and diastolic recovery, with Ca2+ ions shown in yellow and RyR2 peptide in white. Bottom: Schematic representation of Ca2+-dependent RyR2 gating in cardiomyocytes. During diastole, the N-domain of CaM dissociates from RyR2, contributing to the channel’s closed-ready state. Depolarization triggers Ca2+ release through RyR2 (opened/activated state), leading to Ca2+-dependent saturation of the N-domain of CaM, which promotes its rebinding to RyR2, thereby stabilizing the closed/inactivated state essential for limiting Ca2+ leak. As Ca2+ is re-sequestered into the sarcoplasmic reticulum, CaM dissociates from RyR2, restoring the closed-ready state, in preparation for the next excitation cycle. (B) Schematic of the Dynamic-Structure Protein Redesign framework. The schematic illustrates a feedback-loop workflow integrating static (blue) and dynamic (magenta) redesign cycles. The process…

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  Figure 2.. In Silico CaM Redesign Strategy. (A) Static-Structure Redesign. OSPREY 3.0 computational redesign of the CaM N-domain–RyR2 interface identified residues within 7 Å of the RyR2 peptide across nine models. Two positions—S38 and Q41—were consistently predicted as suboptimal, with both Glu and Asp substitutions predicted to improve binding energetics; only the top-ranked variants are shown. Color bar indicates ΔK* energy scores (higher = more favorable). (B) RyR2-CaM complex dynamics. Time-dependent distance between the N-domain residue Cα (S40) and the C-domain residue Cα (E113) demonstrates that wtCaM favors the annealed state (black, low distance), whereas RCaM1 adopts an unlocked conformation (dark blue, high distance). Representative structures below illustrate peptide bending in the unlocked RCaM1 complex versus the straight peptide in wtCaM. The RyR2 peptide is shown in white; CaM N- and C-domains of CaM are highlighted in blue and magenta, respectively. Black and dark blue spheres indicate Cα atoms of residues 40 and 113 in RCaM1 and wtCaM. Ca2+ ions were removed for clarity. Black arrows indicate frames corresponding to annealed states in wtCaM–RyR2 trajectories that were…

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  Figure 3.. In Vitro Validation of the Redesign Strategy. (a) Affinity for the RyR2 peptide at low Ca2+. Steady-state binding of CaM variants to the RyR2 CaM-binding peptide, monitored by intrinsic tryptophan (Trp) fluorescence at sub-saturating Ca2+ (pCa 6.6, ~250 nM). (B) Dissociation rate of RyR2 peptide from CaM variants. Peptide-competition kinetic-exchange assay monitoring Trp fluorescence decay following rapid mixing of preformed RyR2 peptide–CaM complexes with excess spectroscopically silent competitor peptide (NaV1.5). (C) Ca2+ sensitivity of CaM variants. Apparent Ca2+ sensitivity determined from changes in intrinsic Trp fluorescence as Ca2+-CaM binds the RyR2 peptide. (D) Ca2+ dissociation kinetics from CaM bound to RyR2. Stopped-flow analysis of Ca2+ dissociation from Ca2+-saturated CaM–RyR2 complexes. Biphasic Ca2+ release measured with the fluorescent chelator Quin-2, which resolves both the N-domain (fast) and C-domain (slow) release phases. (E-F) Affinity for intact RyR2. FRET-based competition assay measuring binding of CaM variants to intact Ca2+-saturated RyR2 channels. Table (F) summarizes of apparent Ki values, standard errors, and derived parameters from panel E.

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  Figure 4:. Ex vivo Functional Validation of the Redesign Strategy and Mechanistic Basis into CaM–RyR2 Regulation. (a) RCaM2 suppresses pathological Ca2+ leak. Left: schematic illustrating the relationship between CaM binding, RyR2 functional states, and Ca2+ release in the leaky RyR2 model. During diastole, spontaneous Ca2+ release events (leak) reflect defective RyR2 inactivation. In permeabilized cardiomyocytes, RCaM2 exhibits enhanced RyR2 association and is associated with reduced Ca2+ leak frequency. Right: quantification of spontaneous Ca2+ release events (frequency × mass) in permeabilized S2814D ventricular myocytes incubated with wtCaM or RCaM2 (n = 78 cells, mean ± SD, p < 0.05). (B) Model of CaM-mediated RyR2 regulation. Left: schematic illustrating how CaM variants influence the geometry of the RyR2 CaM-binding peptide near residue S3592. wtCaM and RCaM2 are associated with a straighter peptide orientation (~180°), whereas RCaM1 is associated with a bent configuration (~150°) observed in leaky states. Bottom left: Conceptual schematic showing how CaM variants shift RyR2 behavior along the inactivated → closed-ready (leak-prone) continuum. RCaM2 stabilizes the inactivated state,…