Arrhythmia-Associated Calmodulin E105A Mutation Alters the Binding Affinity of CaM to a Ryanodine Receptor 2 CaM-Binding Pocket

Figure 1. Binding interactions of CaMWT and CaME105A mutant with peptide B (RyR2 3584-3602aa) in holo-buffer. Upper panels: Change in power supply to the calorimetric cell during the titration of 200 μM of a peptide B solution into 18 μM of CaMWT (A) and CaME105A (B) at 25 °C in holo-buffer, after the subtraction of the appropriate reference experiments. Lower panels: Integration of the area under each injection, normalized per mol of injectant and plotted as a function of the (peptide)/(CaM) ratio at each point of the CaMWT (C) and CaME105A (D) titrations. Solid red lines represent the non-linear least-square fit of the ITC data to a single-set-of-sites thermodynamic model.

Figure 2. Binding interactions of CaMWT and CaME105A mutant with peptide B (RyR2 3584-3602aa) in apo-buffer. Upper panels: Change in power supply to the calorimetric cell during the titration of 200 μM of a peptide B solution into 18 μM of CaMWT (A) and CaME105A (B) at 25 °C in apo-buffer, after the subtraction of the appropriate reference experiments. Lower panels: Integration of the area under each injection, normalized per mol of injectant and plotted as a function of the (peptide)/(CaM) ratio at each point of the CaMWT (C) and CaME105A (D) titrations. Solid red lines represent the non-linear least-square fit of the ITC data to a single-set-of-sites thermodynamic model.

Figure 3. Binding interactions of CaMWT and CaME105A mutant with peptide F (RyR2 4255-4271aa) in holo-buffer. Upper panels: Change in power supply to the calorimetric cell during the titration of 200 μM of a peptide B solution into 18 μM of CaMWT (A) and CaME105A (B) at 25 °C in holo-buffer, after the subtraction of the appropriate reference experiments. Lower panels: Integration of the area under each injection, normalized per mol of injectant and plotted as a function of the (peptide)/(CaM) ratio at each point of the CaMWT (C) and CaME105A (D) titrations. Solid red lines represent the non-linear least-square fit of the ITC data to a single-set-of-sites thermodynamic model.

Figure 4. Binding interactions of CaMWT and CaME105A mutant with peptide F (RyR2 4255-4271aa) in apo-buffer. Upper panels: Change in power supply to the calorimetric cell during the titration of 200 μM of peptide F solutions into 18 μM of CaMWT (A) and CaME105A (B) at 25 °C in apo-buffer, after the subtraction of the appropriate reference experiments. Lower panels: Integration of the area under each injection, normalized per mol of injectant and plotted as a function of the (peptide)/(CaM) ratio at each point of the CaMWT (C) and CaME105A (D) titrations. Solid red lines represent the non-linear least-square fit of the ITC data to a single-set-of-sites thermodynamic model.

Figure 5. Overlap of the energy-minimized structures of CaMWT (grey ribbon) and CaME105A (blue ribbon) at the EF-hand III binding site. Important residues are shown as stick models, with E105 and A105 colored green and red, respectively, for reasons of emphasis. The model is based on the PDB entry with code 1CLL [52].

Figure 6. Proposed mechanism of RyR2 regulation by CaM. The figure demonstrates the two human RyR2 CaM-binding regions (3584-3602aa and 4255-4271aa), which we propose contribute to a potential intra-subunit RyR2 CaM-binding pocket, essential for CaM/RyR regulation. The distribution of other satellite proteins across structural domains of RyR2 is also presented. CaM, Calmodulin; DR, divergent region; FKBP, calstabin 2; LIZ, leucine–isoleucine zipper; PKA, protein kinase A; PP, protein phosphatase; SPRY; mKAP, PR130; PP1, phosphatase 1; PP2A, phosphatase 2A; RyR2, ryanodine receptor 2 (modified from [53]).