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

Abstract

Pentameric ligand-gated ion channels undergo subtle conformational cycling to control electrochemical signal transduction in many kingdoms of life. Several crystal structures have now been reported in this family, but the functional relevance of such models remains unclear. Here, we used small-angle neutron scattering (SANS) to probe ambient solution-phase properties of the pH-gated bacterial ion channel GLIC under resting and activating conditions. Data collection was optimized by inline paused-flow size-exclusion chromatography, and exchanging into deuterated detergent to hide the micelle contribution. Resting-state GLIC was the best-fit crystal structure to SANS curves, with no evidence for divergent mechanisms. Moreover, enhanced-sampling molecular-dynamics simulations enabled differential modeling in resting versus activating conditions, with the latter corresponding to an intermediate ensemble of both the extracellular and transmembrane domains. This work demonstrates state-dependent changes in a pentameric ion channel by SANS, an increasingly accessible method for macromolecular characterization with the coming generation of neutron sources.

Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel

“…To investigate solution-phase structural properties of DeCLIC in the presence and absence of inhibitory calcium, we collected SANS data in either 10 mM CaCl 2 (Ca 2+ ) or 10 mM EDTA ( apo ) conditions. To optimize for homogeneous monodisperse samples, data were collected from a single peak from an inline size-exclusion column [6]. As calculated by Guinier analysis, the sample radius of gyration (R g ) was slightly larger under Ca 2+ conditions than apo conditions (52.0±0.2 Å compared to 51.6±0.2 Å).…”

“…However, MD simulations were capable of improving goodness of fit to the SANS profile in calcium but not apo conditions, indicating that the available structures-closed and open-did not spontaneously sample conformations representative of the apo time and population average. We have previously reported improved SANS goodness of fits to specific pLGIC states by linear combination of closed and open structures [6]; however, such an approach relies on trusted models of all conformations contributing to the population. Instead, here our simulations failed to support the open X-ray structure as a stable state, leaving open questions as to the physiologically relevant conductive state.…”

“…SEC-SANS experiments [30] were performed at the D22 beamline of Institute Laue-Langevin, using a paused flow approach [6]. In short, the protein sample was loaded on a Superdex 200 Increase 10/300 gel filtration column in-line with the SANS measurement [31,32], exchanging the sample to the D 2 O buffer environment and match-out deuterated DDM (d-DDM) [33] prior to the protein reaching the SANS measuring cell.…”

“…These effects make pLGICs critical targets for a number of therapeutic agents, including anesthetics, benzodiazepines and neurosteroids [4, 5]. A detailed understanding of channel pharmacology and conformational change depends on high-quality structural data, potentially complemented by dynamic information from methods like molecular dynamics (MD) simulations and small-angle scattering [6].…”

Upgraded D22 SEC–SANS setup dedicated to the biology community

“…Other standard proteins measured with better statistics using the flow-rate slowing option are presented in various publications (Johansen et al, 2018;Lycksell et al, 2021;Johansen et al, 2022;Golub et al, 2022;Trewhella et al, 2022).…”