Structural determination of the phosphorylation domain of the ryanodine receptor.
The ryanodine receptor (RyR) is a large, homotetrameric sarcoplasmic reticulum membrane protein that is essential for Ca(2+) cycling in both skeletal and cardiac muscle. Genetic mutations in RyR1 are associated with severe conditions including malignant hyperthermia (MH) and central core disease. One phosphorylation site (Ser 2843) has been identified in a segment of RyR1 flanked by two RyR motifs, which are found exclusively in all RyR isoforms as closely associated tandem (or paired) motifs, and are named after the protein itself. These motifs also contain six known MH mutations. In this study, we designed, expressed and purified the tandem RyR motifs, and show that this domain contains a putative binding site for the Ca(2+)/calmodulin-dependent protein kinase β isoform. We present a 2.2 Å resolution crystal structure of the RyR domain revealing a two-fold, symmetric, extended four-helix bundle stabilized by a β sheet. Using mathematical modelling, we fit our crystal structure within a tetrameric electron microscopy (EM) structure of native RyR1, and propose that this domain is localized in the RyR clamp region, which is absent in its cousin protein inositol 1,4,5-trisphosphate receptor.
Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
Parveen
P
Sharma
Noboru
N
Ishiyama
Usha
U
Nair
Wenping
W
Li
Aiping
A
Dong
Tetsuaki
T
Miyake
Aaron
A
Wilson
Tim
T
Ryan
David H
DH
MacLennan
Thomas
T
Kislinger
Mitsuhiko
M
Ikura
Sirano
S
Dhe-Paganon
Anthony O
AO
Gramolini
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000737:chemistry / Q000378:metabolism; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D020033:Protein Isoforms; D019837:Ryanodine Receptor Calcium Release Channel; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2