National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)
R01 AR068431
United States
American Heart Association
19POST34430178
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
U24 GM116789
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
U24 GM116790
United States
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2022 Title: Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM. Authors: Kavita A Iyer / Yifan Hu / Thomas Klose / Takashi Murayama / Montserrat Samsó / Abstract: Ryanodine receptors (RyRs) are main regulators of intracellular Ca release and muscle contraction. The Y522S mutation of RyR1 causes central core disease, a weakening myopathy, and malignant ...Ryanodine receptors (RyRs) are main regulators of intracellular Ca release and muscle contraction. The Y522S mutation of RyR1 causes central core disease, a weakening myopathy, and malignant hyperthermia, a sudden and potentially fatal response to anesthetics or heat. Y522 is in the core of the N-terminal subdomain C of RyR1 and the mechanism of how this mutation orchestrates malfunction is unpredictable for this 2-MDa ion channel, which has four identical subunits composed of 15 distinct cytoplasmic domains each. We expressed and purified the RyR1 rabbit homolog, Y523S, from HEK293 cells and reconstituted it in nanodiscs under closed and open states. The high-resolution cryogenic electron microscopic (cryo-EM) three-dimensional (3D) structures show that the phenyl ring of Tyr functions in a manner analogous to a "spacer" within an α-helical bundle. Mutation to the much smaller Ser alters the hydrophobic network within the bundle, triggering rearrangement of its α-helices with repercussions in the orientation of most cytoplasmic domains. Examining the mutation-induced readjustments exposed a series of connected α-helices acting as an ∼100 Å-long lever: One end protrudes toward the dihydropyridine receptor, its molecular activator (akin to an antenna), while the other end reaches the Ca activation site. The Y523S mutation elicits channel preactivation in the absence of any activator and full opening at 1.5 µM free Ca, increasing by ∼20-fold the potency of Ca to activate the channel compared with RyR1 wild type (WT). This study identified a preactivated pathological state of RyR1 and a long-range lever that may work as a molecular switch to open the channel.
Entire : Rabbit RyR1 disease mutant Y523S in complex with FKBP12.6 embedde...
Entire
Name: Rabbit RyR1 disease mutant Y523S in complex with FKBP12.6 embedded in lipidic nanodisc in the open state
Components
Complex: Rabbit RyR1 disease mutant Y523S in complex with FKBP12.6 embedded in lipidic nanodisc in the open state
Protein or peptide: Ryanodine receptor 1
Protein or peptide: Peptidyl-prolyl cis-trans isomerase FKBP1B
Ligand: ADENOSINE-5'-TRIPHOSPHATE
Ligand: CALCIUM ION
Ligand: ZINC ION
-
Supramolecule #1: Rabbit RyR1 disease mutant Y523S in complex with FKBP12.6 embedde...
Supramolecule
Name: Rabbit RyR1 disease mutant Y523S in complex with FKBP12.6 embedded in lipidic nanodisc in the open state type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weight
Theoretical: 2.26 MDa
-
Macromolecule #1: Ryanodine receptor 1
Macromolecule
Name: Ryanodine receptor 1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force 2; blot for 1.0s before plunging.
-
Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 11018 / Average exposure time: 3.12 sec. / Average electron dose: 53.19 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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