Dynamic closed states of a ligand-gated ion channel captured by cryo-EM and simulations

Abstract

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high-quality structures in multiple, subtly distinct functional states. However, structural data in this family remain limited, particularly for resting and intermediate states on the activation pathway. Here, we report cryo-electron microscopy (cryo-EM) structures of the proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC) under three pH conditions. Decreased pH was associated with improved resolution and side chain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the β1-β2 and M2-M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryo-EM classes further indicated a low-pH population with an expanded pore. These results allow us to define distinct protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.

Figure 1.. Differential resolution of GLIC cryo-EM structures with varying pH.

(A) Cartoon representations of GLIC, viewed from the membrane plane (top) or from the extracellular side (bottom). Pentameric rings represent the connected extracellular (ECD, light gray) and transmembrane (TMD, medium gray) domains, with the latter embedded in a lipid bilayer (gradient) and surrounding a membrane-spanning pore formed by the second helix from each subunit (M2, dark gray). (A, B) Cryo-EM density for the majority class (state 1) at pH 7 to 4.1 Å overall resolution, viewed as in panel (A) from the membrane plane (top) or from the extracellular side (bottom). Density is colored by local resolution according to scale bar at far right and contoured at both high (left) and low threshold (right) to reveal fine and coarse detail, respectively. (B, C) Density viewed as in panel (B) for state 1 at pH 5, reconstructed to 3.4 Å overall resolution. (B, D) Density as in panel (B) for state 1 at pH 3, reconstructed to 3.6 Å overall resolution.

Figure S1.. Cryo-EM image-processing pipeline.

(A) Representative micrograph from grid screening on a Falcon-3 detector (Talos-Arctica), showing detergent-solubilized GLIC particles. (B) Representative 2D class averages at 0.82 Å/px in a 256 × 256 pixel box and a 180 Å mask. (C) Overview of cryo-EM processing pipelines for data collected at pH 7 (blue), pH 5 (green), and pH 3 (lavender) (see the Materials and Methods section).

Figure S2.. FSC curves and map-to-model FSC for manually modeled cryo-EM reconstructions.

(A) Left: FSC curves for cryo-EM reconstructions at pH 7 before (red) and after (blue) applying a soft mask. Right: FSC curves for cross-validation between model and half-map 1 (light), model and half-map 2 (medium), and model and summed map (dark). (A, B) FSC curves represented as in panel (A) for the dataset at pH 5. (A, C) FSC curves represented as in panel (A) for the dataset at pH 3.

Figure S3.. Distribution of particle orientation for cryo-EM reconstructions.

(A) Cryo-EM map for GLIC at pH 7 (left) and the distribution of orientation sampling after refinement, with low–high values represented by bar height and color (blue–red, right). (A, B) Map and distributions as in panel (A) for the dataset at pH 5. (A, C) Map and distributions as in panel (A) for the dataset at pH 3.

Figure S4.. Cryo-EM densities in α-helical and β-strand regions.

(A) Density (mesh) and corresponding atomic model (sticks, colored by heteroatom) for the M2 helix (E222–E243) at pH 7 (blue, left), pH 5 (green, center), and pH 3 (lavender, right). (A, B) Density and corresponding model, shown as in panel (A), for the β7 strand (P120–I128). Side chains that could not be definitively built at pH 7 (D122, Q124, and L126) are represented by Cβ atoms.

Figure 2.. Side chain rearrangements at subunit interfaces in low-pH structures.

(A) Overlay of predominant (state 1) GLIC cryo-EM structures at pH 7 (blue), pH 5 (green), and pH 3 (lavender), aligned on the full pentamer. Two adjacent subunits are viewed as ribbons from the channel pore, showing key motifs including the β1–β2 and Pro loops and M1–M4 helices from the principal subunit (P), and loop F from the complementary subunit (C). (A, B) Zoom views of the upper gray-boxed region in panel (A), showing cryo-EM densities (mesh at σ = 0.25) and side chain atoms (sticks, colored by heteroatom) around the intersubunit ECD interface between a single principal β1–β2 loop and complementary loop F at each pH. As indicated by dotted circles, side chains including β1–β2 residues K33 and E35 could not be definitively built at pH 7 (left) or pH 5 (center) but were better resolved at pH 3 (right), including a possible hydrogen bond between E35 and T158 (dashed line, 3.2 Å). (A, C) Zoom views of the black-boxed region in panel (A), showing key side chains (sticks, colored by heteroatom) at the domain interface between one principal β1–β2, pre-M1, and M2–M3 region, and the complementary loop-F and M2 region. Dotted circles indicate side chains that could not be definitively built in the corresponding conditions; dashed lines indicate possible hydrogen bonds implicated here in proton-stimulated conformational cycling. Residues contributing to a conserved electrostatic network at the domain interface (D32, R192, Y197) are also shown. (A, D) Zoom views of the lower gray-boxed region in panel (A), showing cryo-EM densities (mesh) and side chain atoms (sticks, colored by heteroatom) around the intersubunit TMD interface between principal and complementary M2–M3 regions at each pH. A potential hydrogen bond between E243 and K248 at pH 7 (left, dashed line, 3.1 Å) is disrupted at pH 5 (center) and pH 3 (right), allowing K248 to reorient towards the subunit interface.

Figure S5.. Local resolution of cryo-EM data processed from equivalent particle subsets.

(A) Density colored by local resolution as in Fig 1B, for the dataset at pH 7. (A, B) Density as in panel (A), generated from a random subset of particles collected at pH 5, equal in number to the pH-7 dataset (86,000). (B, C) Density as in panel (B) from an equivalent subset of particles collected at pH 3.

Figure S6.. Incompatibility of cryo-EM densities with low-pH crystal packing.

Selected region of the crystal lattice of GLIC at low pH (PDB ID 4HFI), with three neighboring molecules (gray) packed in C121 symmetry. Zoom boxes at right show sliced overlays of state-1 reconstructions at pH 7 (blue), pH 5 (green), or pH 3 (lavender) with one crystallographic asymmetric unit (dark gray), revealing clashes of cryo-EM densities with the crystal-lattice neighbor (light gray). Crystal models and cryo-EM densities are shown as surfaces at 6 Å resolution.

Figure S7.. Interfacial rearrangements in previous X-ray structures.

(A) Overlay as in Fig 2A of previous X-ray structures crystallized under resting (white, PDB ID: 4NPQ) and activating (gray, PDB ID: 4HFI) conditions. Two adjacent subunits are viewed as ribbons from the channel pore, showing key motifs including the β1–β2 and Pro loops and M1–M4 helices from the principal subunit (P), and loop F from the complementary subunit (C). (A, B) Zoom views as in Fig 2C of the black-boxed region in panel (A), showing key side chains (sticks, colored by heteroatom) at a single domain interface in resting (white, left) and open (gray, right) X-ray structures. Dotted circle indicates the side chain of K33, which could not be definitively built in resting conditions. Center panel shows major backbone transitions from overlaid resting to open states (orange arrows).

Figure S8.. ECD flexibility in closed-pore simulations.

(A) Root mean-squared deviations over time for Cα-atoms of the ECD (solid) and TMD (dotted) in four replicate 1-μs molecular dynamics simulations of cryo-EM structures determined at pH 7 (blue), pH 5 (green), and pH 3 (lavender). Simulations were performed with side chain charges approximating resting (deprotonated, top) or activating (protonated, bottom) conditions (14). Reference simulations of resting (gray, top) and open (black, bottom) X-ray structures are shown at right. (A, B) Hydration at the hydrophobic gate during simulations under deprotonated (solid) or protonated (striped) conditions as depicted in panel (A), quantified by water occupancy between I233 (I9′) and A237 (A13′) in the channel pore. (B, C) Loop-F root mean-squared deviation during simulations as in panel (B), showing diminished deviations in pH-3 cryo-EM models, and in low- versus neutral-pH X-ray models. (B, C) In panels (B, C), histograms represent median ± 95% confidence interval over all simulations in the corresponding condition.

Figure 3.. Remodeled electrostatic contacts revealed by molecular dynamics.

(A) Zoom views as in Fig 2B of the ECD interface between a single principal (P, right) β1–β2 loop and complementary (C, left) loop F (lavender ribbons) in representative snapshots from molecular dynamics simulations of the pH 3 (state 1) cryo-EM structure, with side chains modified to approximate resting (deprotonated, top) or activating (protonated, bottom) conditions. Depicted residues and proximal ions (sticks, colored by heteroatom) show deprotonated E35 in contact with Na⁺, whereas protonated E35 interacts with T158. (B) Charge contacts between E35 and environmental Na⁺ ions in simulations under deprotonated (solid) but not protonated (striped) conditions of state-1 cryo-EM structures determined at pH 7 (blue), pH 5 (green), or pH 3 (lavender). Histograms represent median ± 95% confidence interval over all simulations in the corresponding condition. Horizontal bars represent median ± confidence interval values for simulations of resting (gray) or open (black) X-ray structures. (B, C) Histograms as in panel (B) showing intersubunit Cα-distances between E35 and T158, which decrease in protonated (striped) versus deprotonated (solid) conditions. (D) Zoom views as in Fig 2D of the TMD interface between principal (P, right) and complementary (C, left) M2–M3 loops (lavender ribbons) in representative snapshots from simulations of the pH 3 (state 1) cryo-EM structure. Depicted residues (sticks, colored by heteroatom) show K248 oriented down towards E243 in deprotonated conditions (top), but out towards the subunit interface in protonated conditions (bottom). (B, E) Histograms as in panel (B) showing electrostatic contacts between E243 and K248, which decrease in pH-3 (lavender) versus pH-7 (blue) and pH-5 structures (green), and in protonated (striped) versus deprotonated (solid) simulation conditions. (F) Principal component (PC) analysis of M2–M3 loop motions in simulations under deprotonated (top) or protonated conditions (bottom) of state-1 cryo-EM structures determined at pH 7 (blue), pH 5 (green), and pH 3 (lavender). For comparison, simulations of previous resting (gray) and open (black) X-ray structures are shown at right, and open-structure results are superimposed in each panel. Inset cartoons illustrate structural transitions associated with dominant PCs (blue–lavender from negative to positive values), representing flipping of residue K248 (PC1) and stretching of the M2–M3 loop (PC2).

Figure S9.. Contraction and untwisting of the ECD in pH-3 state 2.

(A) Views as in Fig 1B of pH-3 state-1 (lavender) and state-2 (purple) cryo-EM densities, shown from the membrane plane (left) or extracellular side (right). Right-hand views are shown for the ECD (above), with purple arrows indicating inward contraction and counter-clockwise untwisting of the ECD in state 2 relative to state 1; and for the TMD (below), with white arrows indicating outward tilting of the upper M2 helices. (B) Histograms indicating parallel trends in ECD contraction (left) and untwisting (right) from resting (gray) to open (black) X-ray structures, and from pH-3 state-1 (lavender) to state-2 (purple) cryo-EM structures. Superimposed error bars show median values ± 25% confidence intervals from molecular dynamics simulations of the three fully built models.

Figure S10.. Cryo-EM image-processing pipeline for minority classes.

(A) Overview of cryo-EM state 2 processing pipeline for data collected at pH 7 (dark blue). The 3D classification with 50 classes was carried out on the largest subset of fairly clean particles (see the Materials and Methods section). (A, B) Same as in (A) but for a dataset at pH 5 (dark green). (A, C) Same as in (A) but for a dataset at pH 3 (dark purple).

Figure S11.. Minority classes at pH 7 and pH 5 display limited differences.

(A) Overlaid side view of backbone ribbons representing two adjacent subunits from GLIC cryo-EM models at pH 7 (light and dark blue), pH 5 (light and dark green), and pH 7 (light gray, PDB ID: 4NPQ) and pH 4 (dark gray, PDB ID: 4HFI). (B) Comparisons of the electrostatic network between the X-ray structures and pH-7 state 1 (blue), state 2 (dark blue), and pH-5 state 1 (green) and state 2 (dark green). The relevant secondary structure elements are labeled. Density of each structure is shown in mesh in their respective color. (C) Pore profiles of cryo-EM static structures (state 1 and state 2) for pH 7 (blue), pH 5 (green) and pH 3 (purple) as well as resting (gray) and open (black) X-ray structures. (D, E, F, G, H) Pore profile of the molecular dynamics derived structures compared to a static structure for resting X-ray, open X-ray Pore profiles of structures (state 1 and state 2) for pH 7 (blue), pH 5 (green), and pH 3 (purple) compared to the molecular dynamics derived structures under activating and resting conditions.

Figure 4.. Minority classes suggest alternative states.

(A) Overlay as in Fig 2A of state-1 (lavender) and state-2 (purple) GLIC cryo-EM structures, along with apparent resting (white, PDB ID: 4NPQ) and open (gray, PDB ID: 4HFI) X-ray structures, aligned on the full pentamer. Adjacent principal (P) and complementary (C) subunits are viewed as ribbons from the channel pore. (A, B) Zoom views of the black-boxed region in panel (A), showing key motifs at the domain interface between one principal β1-β2, pre-M1, and M2–M3 region, and the complementary loop-F and M2 region, for resting (white) and open (gray) X-ray structures overlaid with pH-3 cryo-EM state 1 (top, lavender) or state 2 (bottom, purple). (B, C) Zoom views as in panel (B), showing cryo-EM densities (mesh) and backbone ribbons for pH-3 state 1 (top, lavender) or state 2 (bottom, purple). (D) Pore profiles (38) representing Cα radii for pH-3 cryo-EM state-1 (lavender) and state-2 (purple) structures, open X-ray (black) structure, and quadruplicate 1 μs molecular dynamics simulations of the open X-ray model (median, dashed black; 95% confidence interval, gray).

Figure 5.. Protonation and activation in GLIC pH gating.

(A) Cartoon of the GLIC resting state, corresponding to a deprotonated closed conformation, as represented by the predominant cryo-EM structure at pH 7. Views are of the full protein (top) from the membrane plane, and of the ECD (middle) and TMD (bottom) from the extracellular side, showing key motifs at two opposing subunit interfaces including the principal β1–β2 (green) and M2–M3 loops (blue), complementary F (purple) and β5–β6 (dark gray) loops, and the remainder of the protein in light gray. By the model proposed here, under resting conditions the key acidic residue E35 (green circles) in the β1–β2 loop is deprotonated, and involved in transient interactions with environmental cations (e.g., Na⁺, black circles). Flexibility of the corresponding ECD is indicated by motion lines, associated with relatively low resolution by cryo-EM and high root mean-squared deviation ibn molecular dynamics simulations. In parallel, deprotonated E243 (light blue circles) in the M2 helix attracts K248 (dark blue circles) in the M2–M3 loop, maintaining a contracted upper pore. (A, B) Cartoon as in panel (A), showing a protonated but still closed conformation, as represented by the predominant cryo-EM structure at pH 3. In the ECD, protonation of E35 releases environmental cations and enables it instead to form a stabilizing contact with the complementary subunit via T158 (purple circles) in loop F, associated with partial rigidification of the ECD. In the TMD, protonation of E243 releases K248, allowing it to orient outward/upward towards the subunit/domain interface. (A, C) Cartoon as in panel (A), showing the putative protonated open state, as represented by previous open X-ray structures. Key side chains (E35, T158, E243, and K248) are arranged similar to the protonated closed state, accompanied by general contraction of the ECD including loop F, expansion of the upper TMD including the M2–M3 loop, and opening of the ion conduction pathway.