Three-dimensional localization of divergent region 3 of the ryanodine receptor to the clamp-shaped structures adjacent to the FKBP binding sites.
Of the three divergent regions of ryanodine receptors (RyRs), divergent region 3 (DR3) is the best studied and is believed to be involved in excitation-contraction coupling as well as in channel regulation by Ca(2+) and Mg(2+). To gain insight into the structural basis of DR3 function, we have determined the location of DR3 in the three-dimensional structure of RyR2. We inserted green fluorescent protein (GFP) into the middle of the DR3 region after Thr-1874 in the sequence. HEK293 cells expressing this GFP-RyR2 fusion protein, RyR2(T1874-GFP,) were readily detected by their green fluorescence, indicating proper folding of the inserted GFP. RyR2(T1874-GFP) was further characterized functionally by assays of Ca(2+) release and [(3)H]ryanodine binding. These analyses revealed that RyR2(T1874-GFP) functions as a caffeine- and ryanodine-sensitive Ca(2+) release channel and displays Ca(2+) dependence and [(3)H]ryanodine binding properties similar to those of the wild type RyR2. RyR2(T1874-GFP) was purified from cell lysates in a single step by affinity chromatography using GST-FKBP12.6 as the affinity ligand. The three-dimensional structure of the purified RyR2(T1874-GFP) was then reconstructed using cryoelectron microscopy and single particle image analysis. Comparison of the three-dimensional reconstructions of wild type RyR2 and RyR2(T1874-GFP) revealed the location of the inserted GFP, and hence the DR3 region, in one of the characteristic domains of RyR, domain 9, in the clamp-shaped structure adjacent to the FKBP12 and FKBP12.6 binding sites. COOH-terminal truncation analysis demonstrated that a region between 1815 and 1855 near DR3 is essential for GST-FKBP12.6 binding. These results provide a structural basis for the role of the DR3 region in excitation-contraction coupling and in channel regulation.
Zhang
Jing
J
Cardiovascular Research Group, Department of Physiology, University of Calgary, Alberta T2N 4N1, Canada.
Liu
Zheng
Z
Masumiya
Haruko
H
Wang
Ruiwu
R
Jiang
Dawei
D
Li
Fei
F
Wagenknecht
Terence
T
Chen
S R Wayne
SR
D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D020285:Cryoelectron Microscopy; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D022021:Tacrolimus Binding Proteins; D008274:Magnesium; D002118:Calcium