Ligand discrimination and gating in cyclic nucleotide-gated ion channels from apo and partial agonist-bound cryo-EM structures
- Weill Cornell Medical College, United States
Abstract
Cyclic nucleotide-modulated channels have important roles in visual signal transduction and pacemaking. Binding of cyclic nucleotides (cAMP/cGMP) elicits diverse functional responses in different channels within the family despite their high sequence and structure homology. The molecular mechanisms responsible for ligand discrimination and gating are unknown due to lack of correspondence between structural information and functional states. Using single particle cryo-electron microscopy and single-channel recording, we assigned functional states to high-resolution structures of SthK, a prokaryotic cyclic nucleotide-gated channel. The structures for apo, cAMP-bound, and cGMP-bound SthK in lipid nanodiscs, correspond to no, moderate, and low single-channel activity, respectively, consistent with the observation that all structures are in resting, closed states. The similarity between apo and ligand-bound structures indicates that ligand-binding domains are strongly coupled to pore and SthK gates in an allosteric, concerted fashion. The different orientations of cAMP and cGMP in the 'resting' and 'activated' structures suggest a mechanism for ligand discrimination.
Data availability
The 3 density maps and 3 atomic models have been deposited in PDB under the following accession codes: 6CJQ, 6CJU and 6CJT (coordinates of atomic models), EMD-7482, EMD-7484 and EMD-7483 (density maps).
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apo-bound structure of the prokaryotic SthK channel (coordinates of atomic model)Publicly available at the Electron Microscopy Data Bank (accession no: 6CJQ).
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cAMP-bound structure of the prokaryotic SthK channel (coordinates of atomic model)Publicly available at the Electron Microscopy Data Bank (accession no: 6CJU).
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cGMP-bound structure of the prokaryotic SthK channel (coordinates of atomic model)Publicly available at the Electron Microscopy Data Bank (accession no: 6CJT).
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apo-bound structure of the prokaryotic SthK channel (density map)Publicly available at the Electron Microscopy Data Bank (accession no: EMD-7482).
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cAMP-bound structure of the prokaryotic SthK channel (density map)Publicly available at the Electron Microscopy Data Bank (accession no: EMD-7484).
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cGMP-bound structure of the prokaryotic SthK channel (density map)Publicly available at the Electron Microscopy Data Bank (accession no: EMD-7483).
Article and author information
Author details
Philipp AM Schmidpeter
Funding
National Institute of General Medical Sciences (R01GM124451 and R01GM088352)
- Crina M Nimigean
Deutsche Forschungsgemeinschaft (SCHM 3198/1-1)
- Philipp AM Schmidpeter
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Rheinberger et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Ligand discrimination and gating in cyclic nucleotide-gated ion channels from apo and partial agonist-bound cryo-EM structures
eLife 7:e39775.
https://doi.org/10.7554/eLife.39775
Further reading
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- Structural Biology and Molecular Biophysics
Osmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two networks, referred to here as conserved water networks 1 and 2 (CWN1 and CWN2). Here, we show that PEG400 applied to crystals of dimeric uWNK1 induces de-dimerization. Both the WNK1 the water networks and the chloride-binding site are disrupted by PEG400. CWN1 is surrounded by a cluster of pan-WNK-conserved charged residues. Here, we mutagenized these charges in WNK3, a highly active WNK isoform kinase domain, and WNK1, the isoform best studied crystallographically. Mutation of E314 in the Activation Loop of WNK3 (WNK3/E314Q and WNK3/E314A, and the homologous WNK1/E388A) enhanced the rate of autophosphorylation, and reduced chloride sensitivity. Other WNK3 mutants reduced the rate of autophosphorylation activity coupled with greater chloride sensitivity than wild-type. The water and chloride regulation thus appear linked. The lower activity of some mutants may reflect effects on catalysis. Crystallography showed that activating mutants introduced conformational changes in similar parts of the structure to those induced by PEG400. WNK activating mutations and crystallography support a role for CWN1 in WNK inhibition consistent with water functioning as an allosteric ligand.
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- Structural Biology and Molecular Biophysics
Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates gene expression programs in response to ligand binding. Endogenous and synthetic ligands, including covalent antagonist inhibitors GW9662 and T0070907, are thought to compete for the orthosteric pocket in the ligand-binding domain (LBD). However, we previously showed that synthetic PPARγ ligands can cooperatively cobind with and reposition a bound endogenous orthosteric ligand to an alternate site, synergistically regulating PPARγ structure and function (Shang et al., 2018). Here, we reveal the structural mechanism of cobinding between a synthetic covalent antagonist inhibitor with other synthetic ligands. Biochemical and NMR data show that covalent inhibitors weaken—but do not prevent—the binding of other ligands via an allosteric mechanism, rather than direct ligand clashing, by shifting the LBD ensemble toward a transcriptionally repressive conformation, which structurally clashes with orthosteric ligand binding. Crystal structures reveal different cobinding mechanisms including alternate site binding to unexpectedly adopting an orthosteric binding mode by altering the covalent inhibitor binding pose. Our findings highlight the significant flexibility of the PPARγ orthosteric pocket, its ability to accommodate multiple ligands, and demonstrate that GW9662 and T0070907 should not be used as chemical tools to inhibit ligand binding to PPARγ.
