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Structural basis for diamide modulation of ryanodine receptor

Nature Chemical Biology volume 16, pages 1246–1254 (2020)Cite this article

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Abstract

The diamide insecticide class is one of the top-selling insecticides globally. They are used to control a wide range of pests by targeting their ryanodine receptors (RyRs). Here, we report the highest-resolution cryo-electron microscopy (cryo-EM) structure of RyR1 in the open state, in complex with the anthranilic diamide chlorantraniliprole (CHL). The 3.2-Å local resolution map facilitates unambiguous assignment of the CHL binding site. The molecule induces a conformational change by affecting the S4–S5 linker, triggering channel opening. The binding site is further corroborated by mutagenesis data, which reveal how diamide insecticides are selective to the Lepidoptera group of insects over honeybee or mammalian RyRs. Our data reveal that several pests have developed resistance via two mechanisms, steric hindrance and loss of contact. Our results provide a foundation for the development of highly selective pesticides aimed at overcoming resistance and therapeutic molecules to treat human myopathies.

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Fig. 1: Structures of RyRs in complex with CHL.
Fig. 2: CHL induces the opening of rRyR1.
Fig. 3: Functional analysis of RyR mutations in the CHL binding site.
Fig. 4: FLU binding in DBM RyR.
Fig. 5: CHL effect on RyR1 with CCD mutations.
Fig. 6: CaM regulation of RyR.

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Data availability

The cryo-EM maps and atomic coordinates are deposited in the Protein Data Bank and Electron Microscopy Data Bank, respectively: Ca2+/CHL (PDB 7CF9 and EMD-30343); Ca2+/Caf/ATP/CaM1234 (PDB 6M2W and EMD-30067). Source data are provided with this paper.

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Acknowledgements

We thank Z. Li, J. Xu, X. Jin, S. Yang and X. Zhang from the Instrument Analytical Center of the School of Pharmaceutical Science and Technology at Tianjin University for providing technical support. We acknowledge C. Atkinson and the operating team of the High Resolution Macromolecular cryo-Electron Microscopy facility (HRMEM) at the University of British Columbia (UBC) for data collection. HRMEM is funded by the Canadian Foundation of Innovation, BC Knowledge Development Fund and UBC. We thank A. Li and J. Lu at Nankai University for the maintenance of electron microscopy facilities and user training. This research was enabled in part by support provided by Westgrid (www.westgrid.ca) Cedar cluster and Compute Canada (www.computecanada.ca). This research was funded by the National Key Research and Development Program of China (nos. 2017YFD0201400 and 2017YFD0201403, to Z.Y.), the National Natural Science Foundation of China (no. 31972287, to Z.Y. and Y.L.), CIHR (no. PJT-159601, to F.V.P.), Basis for Supporting Innovative Drug Discovery and Life Science Research from the Japan Agency for Medical Research and Development (no. JP20am0101080j0004, to T.M.), Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 19H03404, to T.M.) and fellowships from the CIHR and Michael Smith Foundation for Health Research (to O.H.-G.).

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Author notes

  1. These authors contributed equally: Ruifang Ma, Omid Haji-Ghassemi, Dan Ma, Heng Jiang.

Authors and Affiliations

  1. Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China

    Ruifang Ma, Dan Ma, Heng Jiang, Lianyun Lin, Li Yao, Arthur Samurkas, Yiwen Wang, Yan Zhang & Zhiguang Yuchi

  2. Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada

    Omid Haji-Ghassemi & Filip Van Petegem

  3. Laboratory of Organic Chemistry, Wageningen University & Research, Wageningen, the Netherlands

    Arthur Samurkas

  4. State Key Laboratory of Elemento-organic Chemistry, Nankai University, Tianjin, China

    Yuxin Li

  5. Key Laboratory of Drug Targets and Drug Leads for Degenerative Diseases, Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, China

    Peng Cao

  6. State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China

    Shian Wu

  7. Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine, Tokyo, Japan

    Takashi Murayama

  8. Interfaculty Institute of Cell Biology, University of Tübingen, Tübingen, Germany

    Bernard Moussian

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Contributions

R.M., O.H.-G. and D.M. performed biochemistry and EM experiments. R.M., H.J., L.Y., Y.L., Y.Z. and T.M. designed and performed cell-based assays. R.M., O.H.-G, L.L., A.S. and P.C. performed modeling and docking experiments. H.J., Y.W., S.W. and B.M. designed and performed Drosophila experiments. Z.Y. and F.V.P. conceived the study and supervised the project. All authors participated in preparation of the manuscript.

Corresponding authors

Correspondence to Filip Van Petegem or Zhiguang Yuchi.

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Extended data

Extended Data Fig. 1 Cryo-EM analysis of rRyR1 in complex with Chlorantraniliprole.

Cryo-EM analysis of rRyR1 in complex with Chlorantraniliprole. a, Elution profile of rRyR1 by gel-filtration chromatography. The left inset shows the plotted standard curve for this column. The molecular weight (MW) estimated from its elution volume is ~ 2540 kDa, suggesting tetrameric form in solution (predicted MW of one subunit is 576.8 kDa). b, A 4–15% gradient SDS-PAGE showing protein marker (PM) in the left lane and purified rRyR1 and FKBP12.6 in the right lane. c, Global reconstruction of the Ca2+/Caf/ATP/CaM1234/CHL dataset and focus refinement mask colored by local resolution from highest resolution (3.2 Å, blue) to lowest resolution (7.2 Å, red). Insets highlight the CHL binding site and local resolution. d, Masked and corrected Fourier shell correlation (FSC) curves for Ca2+/Caf/ATP/CaM1234/CHL dataset calculated from half maps obtained from refinement in RELION (blue), PHENIX map density modified (DM) with Resolve (red), and FSC curved obtained from focused refinement using Mask I (transmembrane mask) as a reference with symmetry expanded particles (gold). e, Similar masked and corrected FSC curves for Ca2+/CHL dataset (green), PHENIX, Resolve density modified and sharpened map (purple), and for the Ca2+/DMSO control dataset (orange).

Extended Data Fig. 2 Data processing pipeline, 3D classification and final global refinements.

Data processing pipeline, 3D classification and final global refinements. a, A low-pass filtered and motion corrected micrograph is shown as an example of the raw data. Particles are highlighted with red boxes. Image was exported from Cryolo boxmanager. 3D classification in this case was done with 6 classes (K = 6). The final reconstruction from RELION along with angular distribution of particles are displayed in the bottom right. Overall resolution is based on FSC cutoff of 0.143. b, Masks used for focused refinements in RELION. (c) and (d) are 3D classification and final global refinement results for the Ca2+/CHL dataset and the Ca2+/DMSO dataset respectively. Left panel shows Euler angular distribution of particles which contributed to the final 3D map. The height and color of bars represent the number of particles at that particular Euler angle with the smallest number in blue and the higher in red. The final corrected masked FSC curves for both Ca2+/CHL and Ca2+/DMSO datasets are displayed, with the FSC cutoff of 0.143 as reference (black dashed lines).

Extended Data Fig. 3 The comparison of the cryo-EM structures from different conditions.

The comparison of the cryo-EM structures from different conditions. a, Comparison of the CHL-binding sites from two conditions: Ca2+/CHL (left) and Ca2+/Caf/ATP/CaM1234/CHL (right). Both maps are contoured at 5 σ. b, EM map (contoured at 4σ) of the side chains involved in CHL coordinating from Ca2+/Caf/ATP/CaM1234/CHL condition. c, The EM maps (contoured at 5.5σ) of the other ligands from the two conditions: Ca2+/CHL (left) and Ca2+/Caf/ATP/CaM1234/CHL (right). d, Left are the side views and bottom views of the pore-forming domains of rRyR1 under Ca2+-only and Ca2+/DMSO conditions. The ion permeation pathway through the transmembrane pore (residues 3639–5037) was illustrated as blue dots (areas accessible to double H2O), green dots (areas accessible to single H2O), and red dots (areas inaccessible to H2O). The hydrophobic gate residue I4937 at the channel gate and G4894 at the selectivity filter (SF) are shown as red and green sticks, respectively. Right is the graph of the pore radii of different rRyR1 structures, with the pore radius plotted against the channel coordinate.

Extended Data Fig. 4 CHL poses in cryo-EM vs docking solutions.

CHL poses in cryo-EM vs docking solutions. a, Comparison of rRyR1-CHL cryo-EM structure (grey) with the top 10 docking solutions (color) using open rRyR1 structure (5TAL) as template. In all induced-fit-docking solutions, it cannot predict the conformational change induced by the binding of CHL. The side chains of Arg4563 in docking solutions clash with the position of CHL observed in our cryo-EM complex structure. b, Comparison of the poses of CHL from the cryo-EM structure (white) and the top docking solution (purple) using rRyR1-CHL cryo-EM structure as template. Docking can reproduce the pose observed in the cryo-EM structure.

Extended Data Fig. 5 Sequence comparison between different species.

Sequence comparison between different species. a, Sequence alignment of the CHL-binding segments. The CHL-coordinating residues are highlighted by green (conserved among all species), orange (conserved among mammals), yellow (conserved among insects), blue (conserved among the Lepidotera), and grey (difference between RyR1 and RyR2). The resistance mutations are indicated by red arrows. b, Phylogenetic analysis of RyRs from different species, including Lepidoptera (Chilo suppressalis RyR: AFN70719.1, Danaus plexippus RyR: OWR50158.1, Spodoptera frugiperda RyR: QCQ29110.1, Mythimna separate RyR: AWV67093.1, Tuta absoluta RyR: APC65631.1, Plutella xylostella RyR: NP_001296002.1), Coleoptera (Leptinotarsa decemlineata RyR: AHW99830.1, Tribolium castaneum RyR: NP_001308588.1), Hymenoptera (Apis mellifera RyR: XP_006569098.1, Ooceraea biroi RyR: XP_026830164.1), Diptera (Drosophila melanogaster RyR: NP_001246211.1, Bactrocera dorsalis RyR: AHY02115.1), Hemiptera (Bemisia tabaci RyR: AFK84957.1, Myzus persicae RyR: XP_022160123.1), Vertebrate (Homo sapiens RyR2: NP_001026.2, Homo sapiens RyR3: NP_001027.3, Xenopus tropicalis RyR1: XP_004917160.1, Danio rerio RyR1: NP_001096041.1, Meleagris gallopavo RyR1: NP_001290128.1, Bos Taurus RyR1: NP_001193706.1, Oryctolagus cuniculus RyR1: NP_001095188.1, Homo sapiens RyR1: NP_000531.2).

Extended Data Fig. 6 Caffeine sensitivity of mutants.

Caffeine sensitivity of mutants. Summary of caffeine potency changes caused by the mutations in CHL-binding site measured by the time-lapse fluorescence assay. # EC50 > 100 mM.

Extended Data Fig. 7 Comparison between RyR1 and RyR2.

Comparison between RyR1 and RyR2. a, Enlarged view of the binding site of CHL-binding site in human RyR2. The non-conserved residues are colored in orange (RyR2) and blue (RyR1). b, Comparison the calmodulin-binding modes in pig RyR2 (Ca2+/Caf/ATP/CaM1234, PDB ID: 6JII) (orange) and rabbit RyR1 (Ca2+/Caf/ATP/CaM1234/CHL) (blue) structures. The C-lobe shows a relatively large conformational change compared to the N-lobe.

Extended Data Fig. 8 Structural and docking analysis of the four resistant mutations.

Structural and docking analysis of the four resistant mutations. a, Induced fit docking analysis shows that the resistance mutations reduce the docking scores for CHL. b–e, Zoomed-in views of the CHL-binding pockets of four RyRs carrying resistance mutations modeled based on rRyR1-CHL complex cryo-EM structure. The sidechains of WT and mutant residues are shown in stick and colored in white and black, respectively. Contacts and clashes between RyR and CHL are indicated by red dash lines and blue solid lines, respectively.

Extended Data Fig. 9 Role of a conserved salt-bridge in pVSD.

Role of a conserved salt-bridge in pVSD. Homologous RyRK4656A actin Cas9 Drosophila (a) shows an abnormal wing structure, which was not observed in WT (b) or heterozygous RyRK4656A/CyO strains (not shown). c, Superposition of rRyR1 in open-state (blue) and closed-state (grey). Arg4563 forms salt bridge with Asp4815 only in the open-state. The S4 moves towards the cytosol upon channel opening. d, Sequence alignment of S1 and S4 helices between rabbit (RAB), DBM RyR, and Drosophila (DRO) reveals that Asp4815 is conserved among all and Arg4653 in rabbit is replaced by a lysine residue in insects. Thus, this salt bridge pair formed in the open-state is conserved.

Extended Data Fig. 10 Schematic representation of RyR modulation by CHL.

Schematic representation of RyR modulation by CHL. Structures under three conditions are compared: 1. activating Ca2+; 2. activating Ca2+ plus ATP and caffeine; 3. activating Ca2+ plus caffeine, ATP, CaM1234 and CHL. The binding of Ca2+ primes the channel, but it mainly stays in the closed states. The binding of ATP and caffeine increases the Po and shift the equilibrium towards the open state. The binding of CHL further increase the Po and stabilizes the open state. The increase in Po correlates with the widening of the channel pore.

Supplementary information

Supplementary Information

Supplementary Tables 1–4.

Reporting Summary

Supplementary Video 1

The binding site of CHL in full-length RyR1.

Supplementary Video 2

The binding of CHL induces channel opening.

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Source Data Fig. 1

Unprocessed gels.

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Ma, R., Haji-Ghassemi, O., Ma, D. et al. Structural basis for diamide modulation of ryanodine receptor. Nat Chem Biol 16, 1246–1254 (2020). https://doi.org/10.1038/s41589-020-0627-5

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