A cryo-EM–based model of phosphorylation- and FKBP12.6-mediated allosterism of the cardiac ryanodine receptor
More flexible with phosphorylation?
The type 2 ryanodine receptor (RyR2) mediates Ca2+ release from the sarcoplasmic reticulum of cardiomyocytes to initiate cardiac muscle contraction. Mutations in this intracellular Ca2+ channel are associated with cardiac diseases that may lead to heart failure. Dhindwal et al. used cryo-EM to determine the structure of rabbit RyR2 in complex with the regulatory protein FKBP12.6 in the closed state at 11.8 Å resolution. They found two conformations of RyR2, which may correspond to the extent of phosphorylation of a domain that harbors several disease-associated mutations. Because the more flexible conformation may correspond to phosphorylated RyR2, the authors suggest that phosphorylation may reduce the energy required for the Ca2+ channel to transition to an open state. These results provide a structural basis for understanding how phosphorylation may affect the activation of RyR2.
Abstract
Type 2 ryanodine receptors (RyR2s) are calcium channels that play a vital role in triggering cardiac muscle contraction by releasing calcium from the sarcoplasmic reticulum into the cytoplasm. Several cardiomyopathies are associated with the abnormal functioning of RyR2. We determined the three-dimensional structure of rabbit RyR2 in complex with the regulatory protein FKBP12.6 in the closed state at 11.8 Å resolution using cryo-electron microscopy and built an atomic model of RyR2. The heterogeneity in the data set revealed two RyR2 conformations that we proposed to be related to the extent of phosphorylation of the P2 domain. Because the more flexible conformation may correspond to RyR2 with a phosphorylated P2 domain, we suggest that phosphorylation may set RyR2 in a conformation that needs less energy to transition to the open state. Comparison of RyR2 from cardiac muscle and RyR1 from skeletal muscle showed substantial structural differences between the two, especially in the helical domain 2 (HD2) structure forming the Clamp domain, which participates in quaternary interactions with the dihydropyridine receptor and neighboring RyRs in RyR1 but not in RyR2. Rigidity of the HD2 domain of RyR2 was enhanced by binding of FKBP12.6, a ligand that stabilizes RyR2 in the closed state. These results help to decipher the molecular basis of the different mechanisms of activation and oligomerization of the RyR isoforms and could be extended to RyR complexes in other tissues.
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Supplementary Material
Summary
Fig. S1. Atomic model of the structure of RyR2-FKBP12.6.
Fig. S2. Degree of phosphorylation of RyR2.
Movie S1. Overall structure of RyR2.
Movie S2. Morph between conformations C1 and C2 for RyR2.
Movie S3. Morph of RyR2 with and without FKBP12.6.
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Information & Authors
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Published In
Science Signaling
Volume 10 | Issue 480
May 2017
May 2017
Copyright
Copyright © 2017, American Association for the Advancement of Science.
Submission history
Received: 26 August 2016
Accepted: 5 May 2017
Acknowledgments
We thank H. Wang and N. Scarsdale for expert computing support. We thank T. Murayama (Juntendo University School of Medicine, Tokyo, Japan) for providing the FKBP12.6 plasmid. Funding: This work was supported by the American Heart Association grant no. 14GRNT19660003 and the Muscular Dystrophy Association grant no. MDA352845. Cryo-EM sample preparation and screening were conducted at the Cryo-Electron Microscope Facility at the Virginia Commonwealth University (VCU), which is supported by the VCU School of Medicine and by Startup Funds to M. Samsó. Direct electron detector data collection was carried out at the Molecular Electron Microscopy Core facility at the University of Virginia (UVA), which is supported by the UVA School of Medicine and NIH grants S10-RR025067 and S10-OD018149. Author contributions: S.D., V.C., D.J.S., and A.R.N. performed the experiments. S.D., K.D., and M.S. collected the data. S.D., J.L., and M.S. analyzed the data. S.D., J.L., and M.S. wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The cryo-EM map of the RyR2 structure was deposited in the 3D Electron Microscopy Data Bank under EMDB ID: 8303. The coordinates of the atomic model for RyR2 structure for conformation 1 were deposited in the Protein Data Bank under PDB ID: 5L1D.
Authors
Funding Information
American Heart Association: award308108, 14GRNT19660003
Muscular Dystrophy Association: award308107, MDA352845
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