title: Conformational dynamics inside amino-terminal disease hotspot of ryanodine receptor.
abstract: The N-terminal region of both skeletal and cardiac ryanodine receptor is a disease mutation hotspot. Recently, a crystal structure of the RyR1 fragment (residues 1-559) was solved. This N-terminal structure contains three separate domains, A, B, and C, and was docked into a central vestibule in a full-length RyR1 cryo-EM map. Here, we reconstructed three-dimensional cryo-EM structures of two GFP-tagged RyR2s with GFP inserted after residue Glu-310 and Ser-437, respectively. The structures of RyR2E310-GFP and RyR2S437-GFP displayed an extra mass on domain B and C, directly validating the predicted docking model. Next, we revealed domain movements in molecular dynamics flexible fitting models in both the closed and open state cryo-EM maps. To further probe the conformational changes, we generated FRET pairs by inserting CFP or YFP in two selected domains, FRET studies of three dual-insertion pairs and three co-expressed single-insertion pairs showed the dynamic structural changes within the N-terminal domains.
authors.affiliation: Department of Physiology and Pharmacology, University of Calgary, Calgary, AB T2N 4N1, Canada; Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB T2N 4N1, Canada.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zhong
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.forename: Xing
authors.initials: X
authors.lastname: Meng
authors.forename: Ruiwu
authors.initials: R
authors.lastname: Wang
authors.forename: Filip
authors.initials: F
authors.lastname: Van Petegem
authors.forename: Terence
authors.initials: T
authors.lastname: Wagenknecht
authors.forename: S R Wayne
authors.initials: SR
authors.lastname: Chen
authors.forename: Zheng
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D002110:Caffeine / Q000737:chemistry; D020285:Cryoelectron Microscopy; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D051379:Mice; D062105:Molecular Docking Simulation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D002110:Caffeine