Interaction between Cardiac Ryanodine Receptor and FK506-Binding Protein Revealed by Cryo-EM and FRET

Type 2 ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. Abnormal calcium release through a dysfunctional RyR2 has been implicated in certain types of sudden cardiac death and heart failure. A 12.6kDa FK506 binding protein (FKBP12.6) tightly associates with RyR2, and stabilizes the close state of RyR2 calcium channel. One proposed mechanism that underlies RyR2 channel dysfunction is the destabilization of the RyR2-FKBP12.6 interaction. In the present study, we mapped the location of green fluorescent protein inserted after residue Tyr-846, near the amino-terminal diseases-causing mutation hotspot, in the three-dimensional (3D) structure of RyR2 by cryo-electron microscopy (cryo-EM). The location of the inserted GFP was found to be close to the previously mapped FKBP12.6 binding site. Based on the structural information that we have learned from 3D cryo-EM, we designed a fluorescence resonance energy transfer (FRET) pair by inserting a yellow fluorescent protein in RyR2 after residue Tyr-846, and attaching a cyan fluorescent protein to FKBP12.6. By monitoring the FRET signals between the donor and acceptor, we are investigating the interaction dynamics between RyR2 and FKBP12.6.Supported by AHA to ZL, NIH to TW, CIHR and HSFA to SRWC.