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.

Structural Modeling of Peptide Toxin - Ion Channel Interactions using RosettaDock

“…In addition, both structural models and native structures represent only snapshots of the full spectrum of protein dynamics. Using static ion channel structures as inputs for molecular dynamics simulations can provide a broader perspective of the dynamics underlying the ion channel gating and modulation (Flood et al, 2019; Galleano et al, 2021; Nguyen et al, 2019; Rovšnik et al, 2021; Suma et al, 2020). Finally, structure-guided computational protein design has achieved significant milestones in recent years (Anishchenko et al, 2021; Cao et al, 2020, 2022; Sahtoe et al, 2022; Silva et al, 2019), and these approaches can be applied to engineer ion channels (Xu et al, 2020; Yang et al, 2016) or design novel ion channel modulators (Xu et al, 2021).…”

Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering

“…Atomic force microscopy images have also revealed a mixture at low pH, including states that predominated under neutral resting conditions ( 24 ). We have also recently used cryo-electron microscopy of GLIC to reconstruct multiple particle classes at pH 3, including a dominant state with an expanded ECD and closed pore, and a minority class intermediate between resting and open crystal structures ( 7 ). Due in part to its low conductance in single-channel recordings ( 35 ), the open probability of GLIC has not been well established.…”

“…The proton-gated cation channel GLIC, from the prokaryote Gloeobacter violaceus , has been a popular model system for structure–function studies in this superfamily ( 3 ); it accounts for 40% of all pLGIC structures in the Protein Data Bank (PDB). This channel is pH gated, and its structure has been solved at both high and low pH in apparent closed and open states, respectively ( 4 – 7 ). A static structural state is considered functionally open if the pore of the structure could conduct relevant ions; otherwise, it is considered a functionally closed state.…”

“…This could suggest some channels (e.g., GLIC) might be able to exist in conformations not yet observed experimentally, or that features such as crystal packing, experimental conditions, or the liquid nitrogen freezing might have biased the conformational distribution. Indeed, the vast majority of GLIC structures to date contain an activated ECD conformation; the few resting-state structures available were reported to notably lower resolution ( 6 , 7 ). Biophysical techniques including electron paramagnetic resonance spectroscopy ( 11 , 23 ) and atomic force microscopy ( 24 ) have suggested GLIC can sample additional states outside the crystalline context, emphasizing the importance of alternative structural approaches to model pLGIC conformational cycling in solution.…”

Illumination of a progressive allosteric mechanism mediating the glycine receptor activation

“…Although capturing structural reorganizations only indirectly, the electrophysiological data suggest that the transition is progressive, with the conformational changes starting from the ECD where the orthosteric site is located and propagating to the TMD to reach the channel gate. Moreover, computational studies of various pLGICs by Molecular Dynamics highlight their passage through a complex conformational landscape with the gating transition being composed of a progressive reorganization of the multimeric receptor architecture [9][10][11][12][13] .…”