Calcium-dependent energetics of calmodulin domain interactions with regulatory regions of the Ryanodine Receptor Type 1 (RyR1)

•Calcium-dependent energies of calmodulin domains binding to RyR1 sites were compared.•CaM domains N and C bind preferentially to RyR13614-3643 rather than RyR11975-1999.•RyR13614-3643 binds (Ca2 +)2-CaMC ~ 5 kcal/mol more favorably than it binds apo CaMC.•CaMN requires Ca2 + to bind RyR1 sites and is ~ 3.5 kcal/mol less favorable than CaMC.•CaM associated with RyR13614-43 retains sequential binding of Ca2 + to its domains.

Calmodulin (CaM) allosterically regulates the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1): apo CaM activates the channel, while (Ca2+)4-CaM inhibits it. CaM-binding RyR1 residues 1975-1999 and 3614-3643 were proposed to allow CaM to bridge adjacent RyR1 subunits. Fluorescence anisotropy titrations monitored the binding of CaM and its domains to peptides encompassing hRyR11975-1999 or hRyR13614-3643. Both CaM and its C-domain associated in a calcium-independent manner with hRyR13614-3643 while N-domain required calcium and bound ~250-fold more weakly. Association with hRyR111975-1999 was weak. Both hRyR1 peptides increased the calcium-binding affinity of both CaM domains, while maintaining differences between them. These energetics support the CaM C-domain association with hRyR13614-3643 at low calcium, positioning CaM to respond to calcium efflux. However, the CaM N-domain affinity for hRyR11975-1999 alone was insufficient to support CaM bridging adjacent RyR1 subunits. Other proteins or elements of the hRyR1 structure must contribute to the energetics of CaM-mediated regulation.

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