Structural mechanism of two gain-of-function cardiac and skeletal RyR mutations at an equivalent site by cryo-EM

Abstract

Mutations in ryanodine receptors (RyRs), intracellular Ca2+ channels, are associated with deadly disorders. Despite abundant functional studies, the molecular mechanism of RyR malfunction remains elusive. We studied two single-point mutations at an equivalent site in the skeletal (RyR1 R164C) and cardiac (RyR2 R176Q) isoforms using ryanodine binding, Ca2+ imaging, and cryo–electron microscopy (cryo-EM) of the full-length protein. Loss of the positive charge had greater effect on the skeletal isoform, mediated via distortion of a salt bridge network, a molecular latch inducing rotation of a cytoplasmic domain, and partial progression to open-state traits of the large cytoplasmic assembly accompanied by alteration of the Ca2+ binding site, which concur with the major “hyperactive” feature of the mutated channel. Our cryo-EM studies demonstrated the allosteric effect of a mutation situated ~85 Å away from the pore and identified an isoform-specific structural effect.

High-resolution structure of the membrane-embedded skeletal muscle ryanodine receptor

“…Although the conformation of the bridging solenoid is conserved, a dramatic shift was found as a result of the improved resolution. The bridging solenoid was resolved at 3.1 Å in the primed state (3.45 Å in the open state), making it possible to assign residues 3,064-3,113 and revealing a shift of up to 50 residues compared with previously published structures (Figure 4; Video S10) (des Georges et al, 2016;Iyer et al, 2020). The shifted region (residues 3,021-3,550) was unassigned in des Georges et al (2016) but was assigned in Iyer et al (2020) with the exception of residues 3,064-3,113.…”

Pathological conformations of disease mutant Ryanodine Receptors revealed by cryo-EM

“…During revision of this manuscript, a study was published looking at the effect of a different disease mutation (R164C) in rabbit RyR1 and its ortholog 41 . In this case, however, the effect of CaM was not considered, and the mutation yielded more subtle changes that did not trigger channel opening (Supplementary Fig 11), suggesting the R164C mutation is milder.…”

Structural and functional interactions between the Ca2+-, ATP-, and caffeine-binding sites of skeletal muscle ryanodine receptor (RyR1)

“…A more flexible conformation on phosphorylation of RyR2 has been suggested to facilitate J o u r n a l P r e -p r o o f the transition to the open state, whereas the binding of FKBP enhanced rigidity and stabilized the RyR2 closed state (28). Malignant hyperthermia-associated RyR1-R615C (29) and RyR1-R164C mutants (27) increased channel openings by affecting interaction between regions in the cytoplasmic shell accompanied by alterations of the Ca 2+ binding site (27). Further, the disease-associated mutations can induce conformational changes to facilitate Ca 2+ binding and increase channel open probability.…”