EPR Structural Dynamics of Calmodulin-RyR Peptide Complex

Abstract

We have used a combination of site-directed spin labeling and EPR spectroscopy to probe the interaction of calmodulin (CaM) with a peptide (RyRp) corresponding to the CaM-binding site on the ryanodine receptor, the muscle calcium release channel. A TOAC spin label, which is bifunctionally and stereospecifically coupled to the alpha-carbon and directly reports peptide backbone dynamics, was used to label RyRp at positions 5, 12, 18, or 25. CW-EPR showed that RyRp alone exhibits a gradient of rotational dynamics (mobility), with the N-terminal region having greater mobility than the C-terminal region. Addition of CaM restricted mobility at all four positions, while preserving the mobility gradient. In the presence of saturating Ca, the immobilization of TOAC-RyRp by CaM is further increased. A bifunctional spin label (BSL) attached stereospecifically at Cys sites i and i+4 was used to label CaM at positions 34 and 38 in the N-lobe and 106 and 110 in the C-lobe. Interspin distances measured by DEER indicated that CaM, in the absence of peptide and Ca, exists in a closed conformation, while Ca induces a shift in equilibrium toward two additional conformations, one more open and another more closed (“compact”). Addition of RyRp caused the two lobes of CaM to collapse toward each other (compact conformation), while exhibiting a broad distribution, indicating high flexibility. Removal of Ca from the complex caused increased distance between the spin labels, indicating greater lobe separation. Distances measured between BSL (on CaM) and TOAC (on RyRp) confirmed the compact but flexible conformation of the complex. These results provide insight into the structural dynamics of the Ca-dependent regulation of RyR by CaM.