Structural dynamics of calmodulin-ryanodine receptor interactions: electron paramagnetic resonance using stereospecific spin labels

Cheng Her,Andrew R Thompson,David D Thomas,Christine B Karim

doi:10.1038/s41598-018-29064-8

Abstract

We have used electron paramagnetic resonance, with rigid and stereospecific spin labels, to resolve structural states in calmodulin (CaM), as affected by binding of Ca and a CaM-binding peptide (RyRp) derived from the ryanodine receptor (RyR), the Ca channel that triggers muscle contraction. CaM mutants containing a pair of cysteines in the N-lobe and/or C-lobe were engineered and labeled with a stereospecifically bound bifunctional spin label (BSL). RyRp was synthesized with and without TOAC (a stereospecifically attached spin-labeled amino acid) substituted for a single amino acid near the N-terminus. Intramolecular DEER distance measurements of doubly-labeled BSL-CaM revealed that CaM exists in dynamic equilibrium among multiple states, consistent with open, closed, and compact structural models. Addition of RyRp shifted the equilibrium partially toward the compact state in the absence of Ca, and completely toward the compact state in the presence of Ca, supporting a conformational selection model. Inter-protein distance measurements show that Ca stabilizes the compact state primarily by inducing ordered binding of the CaM N-lobe to RyRp, while only slightly affecting the C-lobe. The results provide insight into the structural mechanism of CaM-mediated RyR regulation, while demonstrating the power of using two types of rigidly and stereospecifically bound spin labels.

Highlights

We have used electron paramagnetic resonance, with rigid and stereospecific spin labels, to resolve structural states in calmodulin (CaM), as affected by binding of Ca and a CaM-binding peptide (RyRp) derived from the ryanodine receptor (RyR), the Ca channel that triggers muscle contraction
The goal was to select a pair of bifunctional spin label (BSL) labeling sites to distinguish the previously proposed structural models: closed by NMR8, open by X-ray crystallography[4], and compact by X-ray crystallography[15]
We report a unique approach utilizing a combination of BSL, TOAC, and DEER, to resolve and quantitate the structural states of CaM as affected by Ca and RyRp binding

Summary

Introduction

We have used electron paramagnetic resonance, with rigid and stereospecific spin labels, to resolve structural states in calmodulin (CaM), as affected by binding of Ca and a CaM-binding peptide (RyRp) derived from the ryanodine receptor (RyR), the Ca channel that triggers muscle contraction. Intramolecular DEER distance measurements of doubly-labeled BSL-CaM revealed that CaM exists in dynamic equilibrium among multiple states, consistent with open, closed, and compact structural models. The X-ray crystal structure of CaCaM complexed with a peptide constituting the CaM binding site on RyR (CaCaM/RyRp), showed an even more compact structure, in an anti-parallel configuration[15] (Fig. 1c). These high-resolution structures leave significant gaps in our understanding of this important system. It was not possible to compare the structures in that work rigorously with the previously obtained high-resolution structures, because the spin labels used were conventional probes flexibly attached to single Cys residues

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Conclusion