Energetics of Calmodulin Recognition of a Skeletal Muscle Ryanodine Receptor Site

Abstract

In skeletal muscle, the calcium-binding protein calmodulin (CaM) modulates the opening and closing of the ryanodine receptor (RyR1), the main ion channel responsible for calcium release from the sarcoplasmic reticulum during excitation-contraction coupling. By weakly activating RyR1 at low (nM) calcium and inhibiting it at high (µM) calcium, CaM acts as a feedback regulator of calcium levels during muscle contraction. A short region on RyR1 (residues 3614-3640, RyR1p) serves as an anchor for CaM. We investigated the energetics of CaM-RyR1p recognition using Förster resonance energy transfer in auto-fluorescent biosensor constructs (YFP-RyRp-CFP). Using this system, we systematically explored the roles of individual RyR1 residues and each CaM domain at the interface, both in the presence and the absence of calcium. The interplay between the processes of calcium- and target-binding to CaM was probed by quantifying the energy of interaction between wild-type or mutated RyR1p sequences and CaM mutants having non-functional domain-specific Ca2+-binding sites. At low calcium, the interaction is weak (low µM), and mutation Trp3620Ala in the N-terminus of RyR1p (but not mutation Tyr3636Ala in the C-terminus) renders it non-specific. At high calcium, the interaction is at least three orders of magnitude stronger, and CaM C-domain binding to the N-terminal half of RyRp is the main mediator of recognition. A CaM C-domain Ca2+-binding mutant and/or Trp3620Ala RyR1p mutation significantly decrease the binding affinity, while a CaM N-domain Ca2+-binding mutant and/or Tyr3636Ala RyRp mutation have weaker effects. The primary role of CaM C-domain at the interface is maintained in the Tyr3636Ala RyRp complex, whereas Trp3620Ala mutation almost equalizes the energetic contributions of the two CaM domains. Future NMR studies of co-expressed CaM-RyR1p complexes will complement the thermodynamic data with atomic-resolution structural information.