Binding and structural insights of the ryanodine receptor

Ryanodine Receptors (RyR) are large ion channels that are responsible for the release of Ca²⁺ from the sarco/endoplasmic reticulum. The channel consists of a large cytosolic cap which functions as a giant allosteric protein, capable of being modulated by an assortment of binding partners and small molecules. To understand its function and mechanisms one needs to dissect the channel to its smallest parts. Using a combination of isothermal titration calorimetry and x-ray crystallography, two areas have been analyzed: binding by calmodulin (CaM) and the structure of a RyR domain, SPRY2. Calmodulin (CaM) is a Ca²⁺ binding protein that can regulate RyR under conditions of both high and low Ca²⁺ by tuning their Ca²⁺ sensitivity to channel opening and closing in an isoform-specific manner. I analyze the binding of CaM and its individual domains to three different RyR CaM binding regions using isothermal titration calorimetry. I compared binding to skeletal muscle (RyR1) and cardiac (RyR2) isoforms, under both Ca²⁺-loaded and Ca²⁺ free conditions. I find that CaM is able to bind all three regions, but with different binding modes, between the isoforms. Disease mutations target one of the three sites and affect CaM binding and energetics. The SPRY2 domain is one of three repeats of the same fold that are present within the RyR. It has been suggested as a key protein interaction site with dihydropyridine receptors to mediate excitation-contraction coupling in skeletal muscle tissue. RyR1 and RyR2 SPRY2 domains were crystallized and reveal differences with several other known SPRY domain structures. Docking of the RyR1 SPRY2 structure places it in between the central rim and the clamp region. The structure of a disease mutant causing cardiomyopathy is also determined and shows local misfolding. Finally, RyR1 SPRY2 binding to the DHPR II-III loops is undetectable by isothermal titration calorimetry.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat