Localization of the dantrolene-binding sequence near the FK506-binding protein-binding site in the three-dimensional structure of the ryanodine receptor.
Dantrolene is believed to stabilize interdomain interactions between the NH2-terminal and central regions of ryanodine receptors by binding to the NH2-terminal residues 590-609 in skeletal ryanodine receptor (RyR1) and residues 601-620 in cardiac ryanodine receptor (RyR2). To gain further insight into the structural basis of dantrolene action, we have attempted to localize the dantrolene-binding sequence in RyR1/RyR2 by using GFP as a structural marker and three-dimensional cryo-EM. We inserted GFP into RyR2 after residues Arg-626 and Tyr-846 to generate GFP-RyR2 fusion proteins, RyR2Arg-626-GFP and RyR2Tyr-846-GFP. Insertion of GFP after residue Arg-626 abolished the binding of a bulky GST- or cyan fluorescent protein-tagged FKBP12.6 but not the binding of a smaller, nontagged FKBP12.6, suggesting that residue Arg-626 and the dantrolene-binding sequence are located near the FKBP12.6-binding site. Using cryo-EM, we have mapped the three-dimensional location of Tyr-846-GFP to domain 9, which is also adjacent to the FKBP12.6-binding site. To further map the three-dimensional location of the dantrolene-binding sequence, we generated 10 FRET pairs based on four known three-dimensional locations (FKBP12.6, Ser-437-GFP, Tyr-846-GFP, and Ser-2367-GFP). Based on the FRET efficiencies of these FRET pairs and the corresponding distance relationships, we mapped the three-dimensional location of Arg-626-GFP or -cyan fluorescent protein, hence the dantrolene-binding sequence, to domain 9 near the FKBP12.6-binding site but distant to the central region around residue Ser-2367. An allosteric mechanism by which dantrolene stabilizes interdomain interactions between the NH2-terminal and central regions is proposed.
Libin Cardiovascular Institute of Alberta, Department of Physiology & Pharmacology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Ruiwu
R
Wang
Xiaowei
X
Zhong
Xing
X
Meng
Andrea
A
Koop
Xixi
X
Tian
Peter P
PP
Jones
Bradley R
BR
Fruen
Terence
T
Wagenknecht
Zheng
Z
Liu
S R Wayne
SR
Chen
D001665:Binding Sites; D002118:Calcium; D002460:Cell Line; D020285:Cryoelectron Microscopy; D003620:Dantrolene / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D011485:Protein Binding; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins / Q000378:metabolism*
D019837:Ryanodine Receptor Calcium Release Channel; D022021:Tacrolimus Binding Proteins; D003620:Dantrolene; D002118:Calcium
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