Abstract
Inositol trisphosphate receptors (IP3Rs) are ubiquitous Ca2+-permeable channels that mediate release of Ca2+ from the endoplasmic reticulum, thereby regulating numerous processes including cell division, cell death, differentiation and fertilization. IP3Rs are jointly activated by inositol trisphosphate (IP3) and their permeant ion, Ca2+. At high concentrations, however, Ca2+ inhibits activity, ensuring precise spatiotemporal control over intracellular Ca2+. Despite extensive characterization of IP3R, the mechanisms through which these molecules control channel gating have remained elusive. Here, we present structures of full-length human type 3 IP3Rs in ligand-bound and ligand-free states. Multiple IP3-bound structures demonstrate that the large cytoplasmic domain provides a platform for propagation of long-range conformational changes to the ion-conduction gate. Structures in the presence of Ca2+ reveal two Ca2+-binding sites that induce the disruption of numerous interactions between subunits, thereby inhibiting IP3R. These structures thus provide a mechanistic basis for beginning to understand the regulation of IP3R.
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Change history
08 August 2018
In the version of this article initially published, one of the PDB codes associated with the study was listed in the data availability statement as 6DBC but should have been 6DRC. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank M. de le Cruz at the Memorial Sloan Kettering Cancer Center Cryo-EM facility and staff at the New York Structural Biology Simons Electron Microscopy Center for help with data collection, and S.B. Long and T. Walz for comments on the manuscript. This work was supported in part by NIH-NCI Cancer Center Support Grant (P30 CA008748), the Josie Robertson Investigators Program (to R.K.H.) and the Searle Scholars Program (to R.K.H).
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N.P. and R.K.H. designed, performed and analyzed the experiments. N.P. and R.K.H. prepared the manuscript.
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Supplementary Figure 1 Cryo-EM analysis of apo-hIP3R3.
a, Representative raw image and 2D averages of apo hIP3R3. b, Particle selection and focused refinement procedures used to obtain full-channel and focused refinement structures of apo hIP3R3. c, Full-channel reconstruction colored by local resolution estimation from ResMap. d, CD focused refinement reconstruction colored by local resolution estimation from ResMap. e, S1-S4 focused refinement reconstruction colored by local resolution estimation from ResMap. f, FSC plot of full channel (red), CD focused refinement (blue) and S1-S4 focused refinement reconstruction (green). g, FSC plot of full channel reconstruction of all particles compared with the refined model (FSC sum, black), FSC plot of half-map1 compared with the refined model (FSC work, red) and FSC plot of half-map2 compared with the refined model (FSC free, blue) generated by phenix.mtriage.
Supplementary Figure 2 C-terminal domain in apo-hIP3R3.
Cryo-EM density map of apo hIP3R3 lowpass filtered to (a) 3.5 Å and (b) 5.5 Å. Structure of apo hIP3R3 is colored by domain. Boxed region highlights caboxy-terminal domain, which is less well resolved than neighboring regions due to its extensive conformational flexibility.
Supplementary Figure 3 S1′ and S1′′ transmembrane helices in apo-hIP3R3.
a, S1-S4 focused refinement density for S1’, S1” and ordered lipids. One subunit is colored by domain. S1-S4 domain is red, S1’ and S1” are blue and lipid molecules are yellow. Density is contoured at 8σ. b, c, Structural diversity of S1-S2 linker in 6 TM ions channels40,65–68. S1 and S2 colored red and S1-S2 linker colored in blue. Unregistered transmembrane helix in the ryanodine receptor structure is colored magenta. d, Sequence alignment of S1-S2 linker domain of hIP3R1, hIP3R2, hIP3R3 and human type 1 and type 2 ryanodine receptor. 65. Long, S. B., Tao, X., Campbell, E. B. & MacKinnon, R. Atomic structure of a voltage-dependent K+channel in a lipid membrane-like environment. Nature 450, 376–382 (2007). 66. Tang, L. et al. Structural basis for inhibition of a voltage-gated Ca2+ channel by Ca2+ antagonist drugs. Nature 537, 117–121 (2016). 67. Hite, R. K. & MacKinnon, R. Structural Titration of Slo2.2, a Na+-Dependent K+ Channel. Cell 168, 390–399.e11 (2017). 68. Hirschi, M. et al. Cryo-electron microscopy structure of the lysosomal calcium-permeable channel TRPML3. Nature 550, 411–414 (2017).
Supplementary Figure 4 Cryo-EM analysis of IP3-bound hIP3R3.
a, Representative raw image and 2D averages of IP3-bound hIP3R3. b, Initial reference for 3D classification lacking the ARM2 domains and low-pass filtered to 60 Å. c, Particle selection and iterative refinement procedures used to obtain structures of IP3 classes 1, 2, 3, 4 and 5. d, Reconstructions colored by local resolution estimation from ResMap. e, FSC plots of full channel (red) and CD focused refinement (blue) reconstructions. f, FSC plots of all particles compared with the refined model (FSC sum, black), FSC plot of half-map1 compared with the refined model (FSC work, red) and FSC plot of half-map2 compared with the refined model (FSC free, blue) generated by phenix.mtriage.
Supplementary Figure 5 IP3-binding domain.
a-f, IP3-binding site in (a) apo, (b) IP3 class 1, and (c) IP3 class 2, (d) IP3 class 3, (e) IP3 class 4 and (f) IP3 class 5. Coordinating side chains and IP3 are shown as sticks and colored by element. h, Superposition of ligand-free IP3-binding domain structures aligned by BTF1 and BTF2. g, Superposition of IP3-binding domain structures aligned by BTF1 and BTF2. IP3 is shown in sticks.
Supplementary Figure 6 Cryo-EM analysis of Ca2+-bound hIP3R3.
a, Representative size exclusion chromatography traces of hIP3R3 in 5 mM EGTA (red) and 1 mM Ca2+ (blue). b, Representative raw image and 2D averages of Ca2+-bound hIP3R3. c, Particle selection and focused refinement procedures used to obtain full-channel and focused refinement structures of Ca2+-bound hIP3R3. d, Full-channel reconstruction colored by local resolution estimation from ResMap. e, CD focused refinement reconstruction colored by local resolution estimation from ResMap. f, FSC plot of full channel (red), TMD focused refinement (blue) and CD focused refinement reconstructions (black). g, FSC plot of full channel reconstruction of all particles compared with the refined model (FSC sum, black), FSC plot of half-map1 compared with the refined model (FSC work, red) and FSC plot of half-map2 compared with the refined model (FSC free, blue) generated by phenix.mtriage.
Supplementary Figure 7 Ca2+-binding sites in Ca2+-bound hIP3R3.
a-c, Ca2+-binding sites in (a) full channel Ca2+-bound map, (b) CD focused refinement Ca2+-bound map and TMD focused refinement Ca2+-bound map. Coordinating side chains and IP3 are shown as sticks and colored by element. JD Ca2+ is shown as a green sphere and CD Ca2+ is shown as a magenta sphere. d, Superposition of the JD Ca2+ binding domain of Ca2+-bound hIP3R3 (colored by domain) with the Ca2+-binding site of rabbit type 1 ryanodine receptor (grey, PDB: 5T15). The structures are aligned by their Ca2+-coordinating residues. Coordinating residues are shown as sticks and Ca2+ ions are shown as green spheres. Top numbers correspond to hIP3R3 numbering and bottom residues correspond to rabbit type 1 ryanodine receptor numbering. e, Empty Ca2+-binding sites in apo full channel reconstruction.
Supplementary Figure 8 Cryo-EM analysis of low IP3–Ca2+ hIP3R3.
a, Representative raw image and 2D averages of low IP3-Ca2+ hIP3R3. b, Particle selection and focused refinement procedures used to obtain full-channel and focused refinement structures of low IP3-Ca2+ hIP3R3. c, Full-channel reconstruction colored by local resolution estimation from ResMap. d, CD focused refinement reconstruction colored by local resolution estimation from ResMap. e, FSC plot of full channel (red), TMD focused refinement (blue) and CD focused refinement reconstructions (black). f, FSC plot of full channel reconstruction of all particles compared with the refined model (FSC sum, black), FSC plot of half-map1 compared with the refined model (FSC work, red) and FSC plot of half-map2 compared with the refined model (FSC free, blue) generated by phenix.mtriage.
Supplementary Figure 9 Cryo-EM analysis of high IP3–Ca2+ hIP3R3.
a, Representative raw image and 2D averages of high IP3-Ca2+ hIP3R3. b, Particle selection and focused refinement procedures used to obtain full-channel and focused refinement structures of high IP3-Ca2+ hIP3R3. c, Full-channel reconstruction colored by local resolution estimation from ResMap. d, CD focused refinement reconstruction colored by local resolution estimation from ResMap. e, FSC plot of full channel (red), TMD focused refinement (blue) and CD focused refinement reconstructions (black). f, FSC plot of full channel reconstruction of all particles compared with the refined model (FSC sum, black), FSC plot of half-map1 compared with the refined model (FSC work, red) and FSC plot of half-map2 compared with the refined model (FSC free, blue) generated by phenix.mtriage.
Supplementary Figure 10 Features of the high IP3–Ca2+ and low IP3–Ca2+ structures.
Ca2+ and IP3-binding sites in (a) full channel high IP3-Ca2+, (b) TMD focused refinement high IP3-Ca2+, (c) CD focused refinement high IP3-Ca2+, (d) full channel low IP3-Ca2+, (e) TMD focused refinement low IP3-Ca2+ and (f) CD focused refinement low IP3-Ca2+ density maps. Coordinating side chains and IP3 are shown as sticks and colored by element. JD Ca2+ is shown as a green sphere and CD Ca2+ is shown as a magenta sphere.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–10, Supplementary Note and Supplementary Table 1
Supplementary Video 1
Conformational diversity of IP3-bound states in hIP3R3
Supplementary Video 2
Conformational changes between apo state, IP3 class 1 and IP3 class 2
Supplementary Video 3
Conformational changes between apo state, Ca2+-bound state and high IP3-Ca2+ state
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Paknejad, N., Hite, R.K. Structural basis for the regulation of inositol trisphosphate receptors by Ca2+ and IP3. Nat Struct Mol Biol 25, 660–668 (2018). https://doi.org/10.1038/s41594-018-0089-6
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DOI: https://doi.org/10.1038/s41594-018-0089-6
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