Three-dimensional localization of serine 2808, a phosphorylation site in cardiac ryanodine receptor.
Type 2 ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. Phosphorylation of RyR2 by cAMP-dependent protein kinase A and by calmodulin-dependent protein kinase II modulates channel activity. Hyperphosphorylation at a single amino acid residue, Ser-2808, has been proposed to directly disrupt the binding of a 12.6-kDa FK506-binding protein (FKBP12.6) to RyR2, causing a RyR2 malfunction that triggers cardiac arrhythmias in human heart failure. To determine the structural basis of the interaction between Ser-2808 and FKBP12.6, we have employed two independent approaches to map this phosphorylation site in RyR2 by three-dimensional cryo-electron microscopy. In one approach, we inserted a green fluorescent protein (GFP) after amino acid Tyr-2801, and mapped the GFP three-dimensional location in the RyR2 structure. In another approach, the binding site of monoclonal antibody 34C was mapped in the three-dimensional structure of skeletal muscle RyR1. The epitope of antibody 34C has been mapped to amino acid residues 2,756 through 2,803 of the RyR1 sequence, corresponding to residues 2,722 through 2,769 of the RyR2 sequence. These locations of GFP insertion and antibody binding are adjacent to one another in domain 6 of the cytoplasmic clamp region. Importantly, the three-dimensional location of the Ser-2808 phosphorylation site is 105-120 A distance from the FKBP12.6 binding site mapped previously, indicating that Ser-2808 is unlikely to be directly involved in the binding of FKBP12.6 to RyR2, as had been proposed previously.
Meng
Xing
X
Wadsworth Center, New York State Department of Health, Albany, New York 12201, USA.
Xiao
Bailong
B
Cai
Shitian
S
Huang
Xiaojun
X
Li
Fei
F
Bolstad
Jeff
J
Trujillo
Ramon
R
Airey
Judith
J
Chen
S R Wayne
SR
Wagenknecht
Terence
T
Liu
Zheng
Z
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001145:Arrhythmias, Cardiac / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D020285:Cryoelectron Microscopy; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D000939:Epitopes; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012694:Serine; D022021:Tacrolimus Binding Proteins; C509259:FKBP1B protein, mouse; C403484:tacrolimus binding protein 1B