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Mechanism of ligand activation of a eukaryotic cyclic nucleotide−gated channel

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Abstract

Cyclic nucleotide–gated (CNG) channels convert cyclic nucleotide (CN) binding and unbinding into electrical signals in sensory receptors and neurons. The molecular conformational changes underpinning ligand activation are largely undefined. We report both closed- and open-state atomic cryo-EM structures of a full-length Caenorhabditis elegans cyclic GMP−activated channel TAX-4, reconstituted in lipid nanodiscs. These structures, together with computational and functional analyses and a mutant channel structure, reveal a double-barrier hydrophobic gate formed by two S6 amino acids in the central cavity. cGMP binding produces global conformational changes that open the cavity gate located ~52 Å away but do not alter the structure of the selectivity filter—the commonly presumed activation gate. Our work provides mechanistic insights into the allosteric gating and regulation of CN-gated and nucleotide-modulated channels and CNG channel−related channelopathies.

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Fig. 1: Cryo-EM structures of TAX-4 in cGMP-unbound and -bound states.
Fig. 2: The ion permeation pathway of TAX-4 in the closed and open states.
Fig. 3: Comparison of the selectivity filter and central cavity in the closed and open states.
Fig. 4: Functional, structural and computational analyses of F403V V407A mutant TAX-4.
Fig. 5: cGMP binding−induced conformational changes in the CNBD and C-linker.
Fig. 6: cGMP binding−induced conformational changes in the gating ring.
Fig. 7: S6 interactions and cGMP binding−induced conformational changes in S6.

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Data availability

The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-21649 for cGMP-unbound WT TAX-4, EMD-21650 for cGMP-bound WT TAX-4 and EMD-21651 for cGMP-unbound F403V V407A mutant TAX-4. The coordinates of the atomic models have been deposited in the Protein Data Bank under accession numbers PDB 6WEJ for cGMP-unbound WT TAX-4, PDB 6WEK for cGMP-bound WT TAX-4 and PDB 6WEL for cGMP-unbound F403V V407A mutant TAX-4. Source data for Fig. 4a,b and Extended Data Figs. 1b, 3b and 4b are available with the paper online.

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Acknowledgements

We thank S. Tucker and G. Klesse (University of Oxford) for assisting with the installation and use of CHAP and developing a new CHAP module for this work, K. Zhang (Yale University) and Z. Li (Purdue University) for discussion on structure validation, B. Carragher and C. Potter for access to and L. Yen and E. Eng for help with the use of the electron microscopy facility at the New York Structural Biology Center (NYSBC). This work was supported by grants to J.Y. from the National Institutes of Health (NIH) (RO1EY027800 and RO1GM085234), to J.F. from the NIH (RO1GM55440), to G.L. from the National Natural Science Foundation of China (NSFC) (21625302 and 21933010) and to Y.Z. from the NSFC (31700647) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB17000000). Some of the cryo-EM work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at NYSBC, supported by grants from the Simons Foundation (SF349247), NYSTAR and the NIH National Institute of General Medical Sciences (GM103310), with additional support from Agouron Institute (F00316) and NIH (OD019994). Some cryo-EM work was performed at the National Center for CryoEM Access and Training (NCCAT) and the Simons Electron Microscopy Center located at NYSBC, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24 GM129539) and by grants from the Simons Foundation (SF349247) and NY State Assembly Majority.

Author information

Author notes

  1. Minghui Li

    Present address: HIT Center for Life Sciences, School of Life Science and Technology, Harbin Institute of Technology, Harbin, China

  2. These authors contributed equally: Xiangdong Zheng, Ziao Fu and Deyuan Su.

Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, New York, NY, USA

    Xiangdong Zheng, Minghui Li, Huan Li, Shufang Li, Zhengshan Hu, Joachim Frank & Jian Yang

  2. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    Ziao Fu, Shufang Li, Robert A. Grassucci & Joachim Frank

  3. Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, Chinese Academy of Sciences, Kunming, China

    Deyuan Su & Jian Yang

  4. State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

    Yuebin Zhang & Guohui Li

  5. Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA

    Yaping Pan, Zhenning Ren & Ming Zhou

  6. Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Xueming Li

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Contributions

M.L. and J.Y. conceived and initiated the structural project, and X.Z., Z.F., D.S., Y.Z., M.L., X.L., Y.P., M.Z., G.L., J.F. and J. Y. designed experiments, analyzed results and wrote the manuscript. X.Z., D.S. and M.L. performed molecular biological and biochemical experiments. Z.F. and X.Z. performed cryo-EM data acquisition and processing, assisted by R.A.G. and S.L. X.Z. built the atomic models. G.L. designed the molecular dynamics simulation (MDS) strategy, and G.L. and Y.Z. performed MDS. D.S. carried out HEK 293T cell recordings, assisted by H.L. M.Z. supervised and Y.P. performed liposome recordings and analysis, and Z.H. performed supplemental single-channel analysis. H.L. performed confocal imaging, and Z.R. performed liposome flux assays (data not shown). All authors contributed to manuscript discussion, preparation and editing.

Corresponding authors

Correspondence to Guohui Li, Joachim Frank or Jian Yang.

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The authors declare no competing interests.

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Peer review information Peer reviewer reports are available. Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Cryo-EM single-particle analysis of cGMP-unbound TAX-4 reconstituted in lipid nanodiscs.

a, Gel filtration profile of cGMP-unbound TAX-4 nanodisc sample. b, SDS-PAGE of cGMP-unbound TAX-4 nanodisc sample used for cryo-EM. c, A representative motion-corrected micrograph. d, Fourier power spectrum of the micrograph shown in (c). e, Gallery of typical averages from 2D classification. f, Flow chart of cryo-EM image processing. g, Euler angle distribution of particles used in the final 3D reconstruction with C4 symmetry. h, Local resolution of the final density map reconstructed with C4 symmetry. i, Densities of the SF generated from the two half maps, contoured at 5σ and overlaid with the model. j, Gold-standard FSC curves of the final 3D reconstructions with C4 and C1 symmetry, respectively. k, FSC curves for cross-validation between maps and model. Black, model versus the summed map. Blue, model versus the half map that was used for model refinement (called ‘work’). Red, model versus another half map that was not used for model refinement (called ‘free’). Uncropped image for panel b is available as source data.

Source data

Extended Data Fig. 2 Cryo-EM density maps and atomic models of selected key regions in cGMP-unbound and -bound structures.

a, cGMP-unbound. The map was low-pass filtered to 2.6 Å, sharpened with a temperature factor of -117 Å2 and contoured at 5σ. b, cGMP-bound. The map was low-pass filtered to 2.7 Å, sharpened with a temperature factor of -113 Å2 and contoured at 5σ.

Extended Data Fig. 3 Cryo-EM single-particle analysis of cGMP-bound TAX-4 reconstituted in lipid nanodiscs.

a, Gel filtration profile of cGMP-bound TAX-4 nanodisc sample. b, SDS-PAGE of cGMP-bound TAX-4 nanodisc sample used for cryo-EM. c, A representative motion-corrected micrograph. d, Fourier power spectrum of the micrograph shown in (c). e, Gallery of typical averages from 2D classification. f, Flow chart of cryo-EM image processing. g, Euler angle distribution of particles used in the final 3D reconstruction with C4 symmetry. h, Local resolution of the final density map reconstructed with C4 symmetry. i, Densities of the SF generated from the two half maps, contoured at 5σ and overlaid with the model. j, Gold-standard FSC curves of the final 3D reconstructions with C4 and C1 symmetry, respectively. k, FSC curves for cross-validation between maps and model. Black, model versus the summed map. Blue, model versus the half map that was used for model refinement (called ‘work’). Red, model versus another half map that was not used for model refinement (called ‘free’). Uncropped image for panel b is available as source data.

Source data

Extended Data Fig. 4 Cryo-EM single-particle analysis of cGMP-unbound F403V V407A (VA) mutant TAX-4 reconstituted in lipid nanodiscs.

a, Gel filtration profile of cGMP-bound VA TAX-4 nanodisc sample. b, SDS-PAGE of cGMP-bound VA TAX-4 nanodisc sample used for cryo-EM. c, A representative motion-corrected micrograph. d, Fourier power spectrum of the micrograph shown in (c). e, Gallery of typical averages from 2D classification. f, Flow chart of cryo-EM image processing. g, Euler angle distribution of particles used in the final 3D reconstruction with C4 symmetry. h, Local resolution of the final density map reconstructed with C4 symmetry. i, Densities of the SF generated from the two half maps, contoured at 5σ and overlaid with the model. j, Gold-standard FSC curves of the final 3D reconstructions with C4 and C1 symmetry, respectively. k, FSC curves for cross-validation between maps and model. Black, model versus the summed map. Blue, model versus the half map that was used for model refinement (called ‘work’). Red, model versus another half map that was not used for model refinement (called ‘free’). Uncropped image for panel b is available as source data.

Source data

Extended Data Fig. 5 Comparison of the CNBD and C-linker structures of TAX-4 and HCN1.

a,b, Superposition of the CNBD and C-linker structures in unliganded states in stereo view. The α-carbon r.m.s.d is 9.2 Å. c,d, Superposition of the CNBD and C-linker structures in liganded states in stereo view. The α-carbon r.m.s.d is 9.4 Å.

Extended Data Fig. 6 Superposition of S6 of TAX-4 orthologs.

a,b, Superposition of S6 (viewed from the extracellular side) of cGMP-bound (open state) TAX-4, apo (closed state) TAX-4, apo SthK (PDB ID: 6CJQ) and apo LliK (PDB ID: 5V4S), showing the presence of a hydrophobic cavity gate in all the apo structures.

Extended Data Fig. 7 TAX-4 and lipid interactions.

a, Cryo-EM density map of cGMP-unbound TAX-4 reconstituted in lipid nanodiscs. Densities colored in blue are not modeled into TAX-4 and likely represent lipids. b,c, Possible lipid binding in several different regions in the closed state. The extra densities were fit with phosphatidylcholine (lipid 1 and 2) or phosphatidic acid (lipid 3 and 4). d, Cryo-EM density map of cGMP-bound TAX-4 reconstituted in lipid nanodiscs. Densities colored in blue are not modeled into TAX-4 and likely represent lipids. e, Possible lipid binding in the open state. The extra densities were fit with phosphatidylcholine (lipid 1 and 2) or phosphatidic acid (lipid 3). Density maps were contoured at 5σ (b,e) or 3σ (c).

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Supplementary Video 1

Conformational changes produced by cGMP binding and unbinding. Morph of closed- and open-state structures of full-length TAX-4, highlighting the movement of the C-helix of the CNBD, the E′F′ helices of the C-linker, the A′B′ helices of the gating ring, and S4, S5 and S6, viewed parallel to the membrane (side view) and then from the intracellular side (bottom-up view).

Supplementary Video 2

Movement of the S6 hydrophobic cavity gate upon cGMP binding and unbinding. Morph of closed- and open-state structures of S6, highlighting the rotation of F403 and V407 side chains, viewed from the intracellular side (bottom-up view).

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Zheng, X., Fu, Z., Su, D. et al. Mechanism of ligand activation of a eukaryotic cyclic nucleotide−gated channel. Nat Struct Mol Biol 27, 625–634 (2020). https://doi.org/10.1038/s41594-020-0433-5

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