Structural Mapping of Divergent Regions in the Type 1 Ryanodine Receptor Using Fluorescence Resonance Energy Transfer

Ryanodine receptors (RyRs) release Ca2+ to initiate striated muscle contraction. Three highly divergent regions (DRs) in the RyR protein sequence (DR1, DR2, and DR3) may confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryoelectron microscopic (cryo-EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to the FRET acceptors, Cy3NTA or Cy5NTA, targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the “clamp” region of the RyR1 cryo-EM map, which is presumed to interface with Cav1.1. DR1 was localized to the “handle” region, near the regulatory calmodulin-binding site on the RyR. These localizations provide insights into the roles of DRs in RyR allosteric regulation during excitation contraction coupling. [Display omitted] •Divergent regions were localized to RyR1 cryo-EM map using FRET-based trilateration•DR1 is located in the RyR1 handle region, near its Ca2+/CaM-binding site•DR2 is within the RyR1 clamp region, where it may interact with Cav1.1•DR3 is found in the RyR1 clamp region, near the FKBP-binding site Using a FRET-based method, Mahalingam et al. localized three highly variable “divergent region” (DR) sequences within RyR1, a calcium channel that mediates skeletal muscle excitation-contraction coupling. The findings suggest distinct functional roles for each DR, since they occupy three different positions within RyR1.