Modeling a Ryanodine Receptor N-terminal Domain Connecting the Central Vestibule and the Corner Clamp Region

Ryanodine receptors (RyRs) form a class of intracellular calcium release channels in various excitable tissues and cells such as muscles and neurons. They are the major cellular mediators of the release of calcium ions from the sarcoplasmic reticulum, an essential step in muscle excitation-contraction coupling. Several crystal structures of skeletal muscle RyR1 peptide fragments have been solved, but these cover less than 15% of the full-length RyR1 sequence. In this study, by combining modeling techniques with sub-nanometer resolution cryo-electron microscopy (cryo-EM) maps, we obtained pseudo-atomic models for RyR fragments consisting of residues 850-1,056 in rabbit RyR1 or residues 861-1,067 in mouse RyR2. These fragments are docked into a domain that connects the central vestibule and corner clamp region of RyR, resulting in a good match of the secondary structure elements in the cryo-EM map and the pseudo-atomic models, which is also consistent with our previous mappings of GFP insertions by cryo-EM and with FRET measurements involving RyR and FK506-binding protein (FKBP). A combined model of the RyR fragment and FKBP docked into the cryo-EM map suggests that the fragment is positioned adjacent to the FKBP-binding site. Its predicted binding interface with FKBP consists primarily of electrostatic contacts and contains several disease-associated mutations. A dynamic interaction between the fragment and an RyR phosphorylation domain, characterized by FRET experiments, also supports the structural predictions of the pseudo-atomic models.