Abstract
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.
Highlights
High resolution structural information only covers 15% of the full-length sequence of Ryanodine receptors (RyRs)
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-electron microscopy (cryo-EM) map and the pseudo-atomic models, which is consistent with our previous mappings of GFP insertions by cryo-EM and with FRET measurements involving RyR and FK506-binding protein (FKBP)
Multiple Sequences in RyR Involved in FKBP Binding—Marx et al [35] hypothesized that Protein kinase A (PKA) hyper-phosphorylates Ser2808 in RyR2 causing FKBP12.6 (FKBP12) dissociation from RyR2, which directly enhances sarcoplasmic reticulum Ca2ϩ leak and systolic dysfunction in heart failure [36]
Summary
High resolution structural information only covers 15% of the full-length sequence of RyR. It is likely that more than one sequence in RyR participates in FKBP binding, as multiple sequences may form a binding pocket for FKBP when RyR folds and four subunits assemble into a homotetramer In support of this hypothesis, we have mapped two residues, Tyr-846 and Thr-1874, that are widely separated in RyR2’s primary sequence onto the three-dimensional structure by cryo-EM, and we found that the structural domains bearing the two residues are both adjacent to the FKBP-binding site [41, 42]. The fragments docked optimally into a domain that connects the central vestibule and corner clamp region of RyR, which is consistent with our previous results from RyR-GFP mappings by three-dimensional cryo-EM and from FRET measurement of RyR-FKBP interactions [42]. These data suggest that the structural domain formed by the RyR1/RyR2 fragment plays a role in mediating a dynamic domain-domain interaction within RyR and in channel modulation by FKBP
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