Disease Mutations in the Ryanodine Receptor Central Region: Crystal Structures of a Phosphorylation Hot Spot Domain

Abstract

Ryanodine Receptors (RyRs) are huge Ca²⁺ release channels in the endoplasmic reticulum membrane and form targets for phosphorylation and disease mutations. We present crystal structures of a domain in three RyR isoforms, containing the Ser2843 (RyR1) and Ser2808/Ser2814 (RyR2) phosphorylation sites. The RyR1 domain is the target for 11 disease mutations. Several of these are clustered near the phosphorylation sites, suggesting that phosphorylation and disease mutations may affect the same interface. The L2867G mutation causes a drastic thermal destabilization and aggregation at room temperature. Crystal structures for other disease mutants show that they affect surface properties and intradomain salt bridges. In vitro phosphorylation experiments show that up to five residues in one long loop of RyR2 can be phosphorylated by PKA or CaMKII. Docking into cryo-electron microscopy maps suggests a putative location in the clamp region, implying that mutations and phosphorylation may affect the allosteric motions within this area.

Modeling a Ryanodine Receptor N-terminal Domain Connecting the Central Vestibule and the Corner Clamp Region

“…code 4ERT) (17). Both crystal structures have been docked into domain 10, in the corner of the clamp region in the RyR threedimensional cryo-EM map (17,18). Notably, domain 10 is next to the structural domains 9 and 5, where our RyR1 and RyR2 fragments were docked ( Fig.…”

“…The largest peptide fragment, consisting of the first 559 residues, was docked in three structural domains in the center of cytoplasmic assembly (16). Crystal structures of the phosphorylation domain in RyR1 (residues 2,733/2734 -2,940), as well as the corresponding domains in RyR2 (residues 2,699 -2,904) and in RyR3 (residues 2,597-2,800), were recently reported, and they were docked into a region near the corners of the square-shaped cytoplasmic assembly, also known as "clamp" structures (17,18).…”

“…For the RyR1 open state cryo-EM map, the RyR1 fragment model docked in a region that contained both domains 5 and 9, whereas the RyR2 fragment model docked into domain 10, where the phosphorylation domain 2,733/2734 -2,940 was previously docked (supplemental Fig. S3) (17,18). Using the standard linear cross-correlation fitting criterion instead of the Laplacian filter in the rigid-body docking localized both models to the region between domains 5 and 9, the same docking position that was obtained for the RyR1 fragment in the RyR1 open state conformation with the Laplacian filter applied.…”

“…Seven mutant residues in this phosphorylation domain that show direct contacts with the RyR1 fragment model are highlighted with asterisks (in the unstructured loop) or black stick structures. code 4ERT) (17). Both crystal structures have been docked into domain 10, in the corner of the clamp region in the RyR threedimensional cryo-EM map (17,18).…”

“…More recently, crystal structures of a phosphorylation domain in RyR1 (residues 2,733/2734 -2,940) and its corresponding domains in RyR2 (residues 2,699 -2,904) and in RyR3 (residues 2,597-2,800) were reported by two independent research groups. These fragments were docked into a region near each corner of the cytoplasmic assembly (17,18). Nevertheless, further progress by this approach toward a high resolution structure for RyR requires improvement in the cryo-EM resolution of structurally intact RyR and determination of additional fragment crystal structures to fit into the cryo-EM maps.…”