Thermodynamic and Structural Analysis of Calmodulin Interaction with the Skeletal Muscle Ryanodine Receptor

Abstract

In skeletal muscle, the calcium sensor calmodulin (CaM) plays a key role in excitation-contraction coupling, the process of translating neuronal stimuli into mechanical contraction of muscle, by regulating the opening and closing of the calcium channel ryanodine receptor (RyR1). By interacting with this channel differently at high and low calcium, CaM acts as a feedback regulator of calcium levels during muscle contraction: at low calcium, CaM weakly activates RyR1, while calcium-CaM inhibits it. We are investigating the interaction between CaM and its established binding site on RyR1 (CaMBD, residues 3614-3640) using a multi-faceted approach combining biophysical (fluorescence spectroscopy) and structural (solution NMR) methodologies. Forster resonance energy transfer (FRET) experiments in an auto-fluorescent biosensor construct (YFP-CaMBD-CFP) enabled us to determine Gibbs free energies of binding in the absence and the presence of calcium. Using this system, we systematically explored the thermodynamics of molecular recognition between the two biomolecules in high and low calcium environments, as well as the roles played by individual RyR1 residues and each CaM lobe at the interface. To gain additional insights into the interplay between the processes of calcium- and target-binding to CaM, we analyzed the interaction between wild-type or mutated RyR1 CaMBD sequences and CaM mutants in which the calcium-binding sites in one domain had been rendered non-functional. Overall, these experiments show that CaM C-domain binding to molecular determinants in the N-terminus of RyR1 CAMBD dominates the interaction, both in the presence and the absence of calcium. However, the interaction is three orders of magnitude stronger at high calcium levels. To obtain residue-specific information of the binding interface, we are currently undertaking NMR studies of isotopically labeled CaM (wild-type and calcium-binding mutants) in the absence and presence of wild-type and mutated RyR1 sequences.