Structural Insights into Ryanodine Receptor-FKBP12 Interactions in Wild-Type and Mutated States

Abstract

Ryanodine receptors (RyRs) control the release of intracellular calcium from sarcoplasmic reticulum and play a central role in excitation-contraction coupling of skeletal and cardiac muscle. Many modulatory proteins and small molecules interact and regulate the function of RyR, including FKBP12 (FK506 binding proteins of 12), a protein ligand that binds RyR with nanomolar affinity. FKBP12 stabilizes the closed state of the channel and reduces the appearance of subconductance states. Dissociation of FKBP12 can cause the calcium leak from RyR and has been implicated in several disorders, such as muscular dystrophy, sarcopenia, heart failure, diabetes, or Alzheimer's. Previous studies using cryo-EM and FRET suggest FKBP12 binds to the periphery of cytoplasmic domain, but the exact binding determinants within the RyR protein at the single amino acid level had remained unclear. The recent high-resolution cryo-EM studies also disagree on the identity of the RyR domain interacting with FKBP12. We determined the high-resolution crystal structures of several RyR domains. Computational docking of these structures, combined with FRET trilateration experiments, show that SPRY1 domain is located next to FKBP12. The key residues in SPRY1 critical for the binding of FKBP12 were identified by molecular dynamics flexible fitting and verified by mutagenesis and functional assays. Disease mutations in SPRY1 were investigated by x-ray crystallography. One of them directly affects FKBP12 binding properties by interfering domain folding. This structural model provides a valuable template for designing therapeutic drugs targeting RyR-FKBP12 interface to treat the diseases mentioned above.