Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM

Abstract

Ryanodine receptors (RyRs) are main regulators of intracellular Ca 2+ 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 2+ activation site. The Y523S mutation elicits channel preactivation in the absence of any activator and full opening at 1.5 µM free Ca 2+ , increasing by ∼20-fold the potency of Ca 2+ 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.

Interplay between Mg2+and Ca2+at multiple sites of the ryanodine receptor

“…While the conformational change induced by CAS occupancy in the close vicinity may help positioning the EF hand domain, the movement of the EF hand is of larger magnitude, suggesting direct interaction of the cation at the EF hand loop(s). This was well documented for the activated conformation induced by µM Ca 2+ , showing rotation and translation of the EF hand with respect to the CD (9.7º and 6.2 Å versus 2.9º and 1.3 Å, respectively) (33). In the inactivated conformation induced by mM Ca 2+ the EF hand domain moved further (Fig.…”

“…10) that abates its angle by 6º when RyR1 is stimulated with Ca 2+ (33). It is likely that this lever forms part of the long-range allosteric pathway connecting DHPR and RyR1's channel, as a severe RyR1 disease mutation in the DHPR-facing region of the lever abated the angle of this lever by 5º under closed-state conditions (33) and increased the sensitivity of DHPR to depolarization by 40 mV (40). Thus, the change in the DHPR voltage sensor upon depolarization, potentially moving (abating) the long levers of RyR1, could constitute a mechanism to directly induce a separation of the inner helices, disrupting the Mg 2+ coordination and unblocking the channel.…”

Interplay between Mg2+ and Ca2+ at multiple sites of the ryanodine receptor

“…One potential mechanism is via a ~100 Å-long lever that spans from the DHPR-facing region of RyR1 to the base of the CD (Fig. 10 ) that abates its angle by 6° when RyR1 is stimulated with Ca 2+ 33 . It is likely that this lever forms part of the long-range allosteric pathway connecting DHPR and RyR1’s channel, as a severe RyR1 disease mutation in the DHPR-facing region of the lever abated the angle of this lever by 5° under closed-state conditions 33 and increased the sensitivity of DHPR to depolarization by 40 mV 40 .…”

The Effect of Acidic Residues on the Binding between Opicalcin1 and Ryanodine Receptor from the Structure–Functional Analysis

“…All RyRs models were downloaded from the Protein Data Bank (PDB) () . We selected RyRs in the open state as the receptor (PDB ID: RyR1, 7T65; RyR2, 7U9Z ) for molecular docking, and the ligand structure was predicted by AlphaFold2 . The peptide with acid residue site mutation to alanine was used for spatial structure prediction, and the peptide with the highest parameter score was selected as the best prediction .…”