Ligand discrimination and gating in cyclic nucleotide-gated ion channels from apo and partial agonist-bound cryo-EM structures
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
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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Further reading
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- Structural Biology and Molecular Biophysics
The KCNH family of potassium channels serves relevant physiological functions in both excitable and non-excitable cells, reflected in the massive consequences of mutations or pharmacological manipulation of their function. This group of channels shares structural homology with other voltage-gated K+ channels, but the mechanisms of gating in this family show significant differences with respect to the canonical electromechanical coupling in these molecules. In particular, the large intracellular domains of KCNH channels play a crucial role in gating that is still only partly understood. Using KCNH1(KV10.1) as a model, we have characterized the behavior of a series of modified channels that could not be explained by the current models. With electrophysiological and biochemical methods combined with mathematical modeling, we show that the uncovering of an open state can explain the behavior of the mutants. This open state, which is not detectable in wild-type channels, appears to lack the rapid flicker block of the conventional open state. Because it is accessed from deep closed states, it elucidates intermediate gating events well ahead of channel opening in the wild type. This allowed us to study gating steps prior to opening, which, for example, explain the mechanism of gating inhibition by Ca2+-Calmodulin and generate a model that describes the characteristic features of KCNH channels gating.
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- Structural Biology and Molecular Biophysics
The M2 proton channel aids in the exit of mature influenza viral particles from the host plasma membrane through its ability to stabilize regions of high negative Gaussian curvature (NGC) that occur at the neck of budding virions. The channels are homo-tetramers that contain a cytoplasm-facing amphipathic helix (AH) that is necessary and sufficient for NGC generation; however, constructs containing the transmembrane spanning helix, which facilitates tetramerization, exhibit enhanced curvature generation. Here, we used all-atom molecular dynamics (MD) simulations to explore the conformational dynamics of M2 channels in lipid bilayers revealing that the AH is dynamic, quickly breaking the fourfold symmetry observed in most structures. Next, we carried out MD simulations with the protein restrained in four- and twofold symmetric conformations to determine the impact on the membrane shape. While each pattern was distinct, all configurations induced pronounced curvature in the outer leaflet, while conversely, the inner leaflets showed minimal curvature and significant lipid tilt around the AHs. The MD-generated profiles at the protein–membrane interface were then extracted and used as boundary conditions in a continuum elastic membrane model to calculate the membrane-bending energy of each conformation embedded in different membrane surfaces characteristic of a budding virus. The calculations show that all three M2 conformations are stabilized in inward-budding, concave spherical caps and destabilized in outward-budding, convex spherical caps, the latter reminiscent of a budding virus. One of the C2-broken symmetry conformations is stabilized by 4 kT in NGC surfaces with the minimum energy conformation occurring at a curvature corresponding to 33 nm radii. In total, our work provides atomistic insight into the curvature sensing capabilities of M2 channels and how enrichment in the nascent viral particle depends on protein shape and membrane geometry.