Conformational Dynamics inside Amino-Terminal Disease Hotspot of Ryanodine Receptor

The N-terminal region of both skeletal and cardiac ryanodine receptor is a disease mutation hotspot. Recently, a crystal structure of the RyR1 fragment (residues 1–559) was solved. This N-terminal structure contains three separate domains, A, B, and C, and was docked into a central vestibule in a full-length RyR1 cryo-EM map. Here, we reconstructed three-dimensional cryo-EM structures of two GFP-tagged RyR2s with GFP inserted after residue Glu-310 and Ser-437, respectively. The structures of RyR2E310-GFP and RyR2S437-GFP displayed an extra mass on domain B and C, directly validating the predicted docking model. Next, we revealed domain movements in molecular dynamics flexible fitting models in both the closed and open state cryo-EM maps. To further probe the conformational changes, we generated FRET pairs by inserting CFP or YFP in two selected domains, FRET studies of three dual-insertion pairs and three co-expressed single-insertion pairs showed the dynamic structural changes within the N-terminal domains. [Display omitted] •Two cryo-EM 3D reconstructions validate the docking model of RyR N-terminal domains•Docking models in the closed and open states predict N-terminal domain movements•FRET analysis reveals dynamic conformational changes within RyR N-terminal domains Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca2+ from the sarcoplasmic reticulum and impact the work of skeletal and cardiac muscle. Zhong et al. combine cryo-EM, FRET, and molecular dynamics to characterize the structure and conformational dynamics inside the N-terminal domains.