Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

Figures

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  FIGURE 1. Tertiary and quaternary structure of CaM variants in complex with RyR1/2 peptides. (A) Cartoon representation of CaM in three different conformational states, namely apo (PDB: 1DMO (Zhang et al., 1995)), Ca2+-bound (PDB: 1CLL (Chattopadhyaya et al., 1992)) and complexed with the RyR2 peptide used in this study (PDB: 6Y4O (Holt et al., 2020)). The N-terminal domain is colored in yellow, the linker region in grey, while the C-terminal domain in green. The RyR2 peptide is represented in magenta. Ca2+ ions are represented as red spheres while the side chains of N97 and Q135 are represented in sticks with C atoms colored according to the structural region, O atoms in red and N atoms in blue. (B) Pairwise sequence alignment of the Calmodulin Binding Domain-2 of human RyR1 (Uniprot entry P21817) and RyR2 (Uniprot entry Q92736). The sequence relative to the two RyR peptides employed in this study is highlighted in yellow, the residues that are not identical in such region are represented in bold and colored in red. (C) Near-UV CD spectra (250–320 nm) of 50 μM CaM were collected in the presence of 500 µM EGTA (black dashed line) and after sequential additions of 1 mM Ca2+ (black solid…

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  FIGURE 2. Investigation of secondary structure of CaM variants and their RyR1/2 peptide complexes. Far-UV CD spectra (200–250 nm) of 10 μM CaM variants alone (top panels, black), and incubated with 20 μM RyR1 (center panels, blue), or 20 μM RyR2 (bottom panels, red), were collected in the presence of 300 µM EGTA (dashed lines) and after the addition of 600 µM Ca2+ (300 µM free Ca2+, solid lines). The spectrum of sole buffer was considered as blank and subtracted; each curve represents the average of five accumulations. Temperature was set at 25°C.

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  FIGURE 3. Limited proteolysis of CaM variants. CaM variants (26.6 µM) were incubated with .4 µM trypsin (ratio 1:66 trypsin: CaM) in the presence of 2 mM EGTA (A) or Ca2+ (B). Reactions were run for 10 min at 25°C and then blocked by boiling samples for 10 min at 96°C. Proteolytic fragments were loaded on a 15% SDS-PAGE and Coomassie blue-stained. For each investigated condition, the untreated protein was loaded as a control.

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  FIGURE 4. CaM affinity for RyR1 and RyR2 peptides assessed by fluorescence spectroscopy. (A) One micromolar RyR1 (left column) or RyR2 (right column) peptide was incubated with increasing amounts of WT (top row), N97I (middle row) and Q135P (bottom row) CaM in the presence of 100 µM Ca2+. Data are reported as a function of the peptide fraction bound to CaM (see Materials and Methods for details). Curves report the mean ± std of each point obtained in three technical replicas. Representative fitting to one-site saturation ligand binding curve is superposed to each titration set. (B) Scatter plot of replicates reporting the KD values calculated from fitting procedures in each individual dataset using a one-site binding model. Stars represent t-test statistical significance: * p-value ≤ 0.05, ** p-value ≤ 0.01.

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  FIGURE 5. Thermodynamics of CaM-RyR1/2 peptide interaction assessed by isothermal titration calorimetry. (A) Examples of ITC titration curves obtained for each CaM variant upon interaction with RyR1 or RyR2 peptides. Measurements were performed at 25°C using 20 mM Tris pH 7.5, 150 mM KCl, 5 mM Ca2+ as working buffer and setting stirring at 750 rpm. Each titration consisted in thirty 1-µL injections of 125 µM RyR1 or RyR2 (into the titrant syringe) with 10 µM CaM variants. (B) Scatter plot of replicates summarizing the KD values calculated from the fitting using a one-site binding model (see Materials and Methods). Stars represent the p-values: *p ≤ 0.05, ***p ≤ 0.001. Data for WT CaM titration with RyR2 are from (Dal Cortivo et al., 2022).

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  FIGURE 6. Kinetics of CaM-RyR1/2 peptide interaction investigated by surface plasmon resonance. (A) Sensorgrams collected by flowing different amounts of RyR1 and RyR2 (125 nM—2 µM) on immobilized His-CaM variants using 20 mM Tris pH 7.5, 150 mM KCl, 0.005% Tween 20, 5 mM Ca2+, 100 µM DTT as a running buffer. Association and dissociation phases were followed for 60 s and 300 s, respectively. Experimental curves (black solid lines) are shown together with theoretical curves (red or blue solid lines) according to a 1:1 Langmuir binding model; fitting for association and dissociation phases led to the rate constants (k on and k off) reported in each panel (mean ± s.e.m. of 8-20 independent binding curves). (B) Scatter plot of replicates and statistical analysis comparing rate constants for WT and each pathogenic CaM variant. Stars represent p-values: *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

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  FIGURE 7. Backbone flexibility of CaM-RyR1/2 complexes. Cα-Root-Mean Square Fluctuation (RMSF) of CaM (top panels) and RyR1 or RyR2 peptides (bottom panels) calculated over 1.2 μs MD simulations of the respective complex (WT: black, N97I: blue, and Q135P: red).

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  FIGURE 8. Effects of CaM variants on the connectivity of hub residues of CaM (top panels) and RyR1/2 peptides (bottom panels). Hubs were defined as residues with degree ≥6 in the Protein Structure Network (PSN) of at least one variant. ∆Degree is calculated as the difference in hub degree between the variant (N97I: black, Q135P: red) and the WT.

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  FIGURE 9. Robustness of intramolecular communication between EF-hands in CaM-RyR1/2 complexes. (A) Intramolecular communication among EF-hands in the CaM-RyR1 (left) and CaM-RyR2 (right) complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM WT (black), N97I (red), and Q135P (green) variants in complex with RyR peptides. (B) Effects of specific RyR peptides on the intramolecular communication among EF-hands in CaM-RyR complexes. Communication robustness between EF-hands (EF1 to EF4, represented by their bidentate Glu residues) in CaM variants in complex with RyR1 (black) and RyR2 (red) peptides.