Thermodynamic Analysis of Calmodulin Recognition of the Ion Channel Ryanodine Receptor

Abstract

In skeletal muscle, the calcium binding protein calmodulin (CaM) plays an essential role in excitation-contraction coupling by modulating the opening and closing of the calcium channel ryanodine receptor (RyR1). Biochemical studies have mapped the CaM binding site (CaMBD) to a short region on RyR1 comprising residues 3614-3643. By interacting with this region differently at high and low calcium, CaM acts as a 'switch', providing essential feedback in Ca2+ levels during muscle contraction. Our X-ray structure of Ca2+-CaM bound to RyR1 CaMBD (2BCX) revealed a novel '1-17' motif of RyR1 hydrophobic amino-acids anchoring the CaM domains. In order to gain additional insights into the CaM-RyR1 interaction, we are pursuing thermodynamic studies of CaM-RyR1 CaMBD complexes at several Ca2+ concentrations.The energy of interaction between CaM and RyR1 CaMBD was determined using Forster resonance energy transfer in an auto-fluorescent biosensor construct (YFP-CaMBD-CFP). Fluorescence titrations at increasing concentrations of CaM enabled us to determine association constants and free energies of binding at high and low calcium. Interestingly, the affinities of apo- and Ca2+-CaM for RyR1 CaMBD differed by orders of magnitude (micromolar vs. sub-nanomolar). Mutational analysis was performed for RyR1 residues W3620 and F3636 which form close contacts with the CaM C- and N-domains in the 2BCX complex. Both in the presence and absence of calcium, mutation of W3620 to alanine resulted in significant effects on the binding affinity, while the F3636 mutation had weaker effects. Titrations with the individual CaM domains and CaM calcium-binding mutants show that the CaM C-domain is the main mediator of interaction. Future studies will further explore residue-specific differences in CaM-RyR1 recognition and the interplay between the processes of calcium- and target-binding to CaM.Funding: Drake University, NIH R01 GM57001, University of Iowa FUTURE in Biomedicine program.