Alterations in calmodulin-cardiac ryanodine receptor molecular recognition in congenital arrhythmias

Abstract

Calmodulin (CaM), a ubiquitous and highly conserved Ca²⁺-sensor protein involved in the regulation of over 300 molecular targets, has been recently associated with severe forms of lethal arrhythmia. Here, we investigated how arrhythmia-associated mutations in CaM localized at the C-terminal lobe alter the molecular recognition with Ryanodine receptor 2 (RyR2), specifically expressed in cardiomyocytes. We performed an extensive structural, thermodynamic, and kinetic characterization of the variants D95V/H in the EF3 Ca²⁺-binding motif and of the D129V and D131H/E variants in the EF4 motif, and probed their interaction with RyR2. Our results show that the specific structural changes observed for individual CaM variants do not extend to the complex with the RyR2 target. Indeed, some common alterations emerge at the protein-protein interaction level, suggesting the existence of general features shared by the arrhythmia-associated variants. All mutants showed a faster rate of dissociation from the target peptide than wild-type CaM. Integration of spectroscopic data with exhaustive molecular dynamics simulations suggests that, in the presence of Ca²⁺, functional recognition involves allosteric interactions initiated by the N-terminal lobe of CaM, which shows a lower affinity for Ca²⁺ compared to the C-terminal lobe in the isolated protein.

Keywords: Allostery; Arrhythmia; Calcium; Calmodulin; Molecular dynamics.

Fig. 1

Structural analysis of CaM variants under different conditions. a Structural representation of CaM in the apo form (left, PDB: 1DMO [40]), Ca²⁺-bound (middle, PDB: 1CLL [41]) and complexed with RyR2 peptide (right, PDB: 6Y4O [15]). EF-hand motifs and RyR2 peptide (in pink) are represented as cartoon, Ca²⁺ ions are represented as red spheres. b Near UV CD spectra of 50 μM CaM variants in the presence of 0.5 mM EGTA (black lines) and after sequential additions of 1 mM Ca²⁺ (red lines) and 100 μM RyR2 (green lines)

Fig. 2

Bidimensional NMR spectra of CaM variants. a Three-dimensional structure of Ca²⁺-CaM (PDB entry: 1CLL [41]). Mutated residues D95, D129 and D131 are shown as cyan sticks on the protein structure. b–f Overlay of the ¹H-¹⁵ N HSQC NMR spectra of 100 µM ¹⁵ N-WT CaM (red) and its variants in the presence of 2 mM Ca²⁺ ions

Fig. 3

Structural effects of CaM-RyR2 interaction monitored by NMR spectroscopy. a Three-dimensional structure of Ca²⁺-loaded CaM in complex with RyR2 peptide (PDB: 6Y4O [15]). Mutated residues D95, D129 and D131 are shown as sticks. b–d Overlay of the 2D ¹H-¹⁵ N HSQC NMR spectra of ¹⁵ N-WT CaM and its variants in presence of 2 mM Ca²⁺ ions and 2 eq. of RyR2 peptide

Fig. 4

Surface Plasmon Resonance kinetic analysis of CaM-RyR2 interaction. a Example of titration with increasing concentrations of RyR2 (165 nM to 2.1 μM) injected on WT His-CaM immobilized on a His-Cap sensor chip. Association (grey box) and dissociation (orange box) phases were followed for 60 s and 300 s, respectively. Association and dissociation rate constants (k^on and k^off) are reported in each panel. Experimental curves (black solid lines) are shown together with theoretical curves (red solid lines) after fitting to a 1:1 Langmuir binding model

Fig. 5

Protein-peptide Structure Network analysis based on Molecular Dynamics simulations. a–f High degree hubs of WT CaM-RyR2 complex and CaM variants D95H, D95V, D131E, D131H and D129V. Protein structure is shown as tubes, with CaM colored in grey and RyR2 peptide in yellow. Ca²⁺ ions are shown as black spheres. The Cα of hub residues with degree 6, 7 and 8 are represented as blue, green and orange spheres, respectively, with radius proportional to the degree of the hub. Residues with mutations associated with cardiac arrhythmias are labelled in red. Hub residues belonging to the RyR2 peptide are framed. g Diagram representing the most robust communication pathways connecting Glu residues, which constitute the bidentate Ca²⁺-coordinators of the 4 EF-hands in WT and mutated CaM-RyR2 complexes. EF1 is shown in yellow, EF2 in green, EF3 in blue and EF4 in orange. Blue arrows: communication robustness index (CR) > 0.1, green arrows: CR > 0.2, yellow arrows: CR > 0.3, red arrows: CR > 0.4 (individual values are reported in Fig. S16). Cartoon representation of the 3D structure of Ca²⁺-loaded CaM-RyR2 complex where proteins structure is represented as cartoon and, with EF1 colored in yellow, EF2 in green, EF3 in orange, EF4 in cyan and RyR2 peptide in pink. Ca²⁺ ions are shown as red spheres; the solvent-accessible surface of CaM-RyR2 complex is shown in transparency and colored according to each EF-hand