The Free Energy Landscape of GABA Binding to a Pentameric Ligand-Gated Ion Channel and Its Disruption by Mutations.

Federico Comitani,Carla Molteni,Vittorio Limongelli

doi:10.1021/acs.jctc.6b00303

Abstract

Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop superfamily are important neuroreceptors that mediate fast synaptic transmission. They are activated by the binding of a neurotransmitter, but the details of this process are still not fully understood. As a prototypical pLGIC, here we choose the insect resistance to dieldrin (RDL) receptor involved in resistance to insecticides and investigate the binding of the neurotransmitter GABA to its extracellular domain at the atomistic level. We achieve this by means of μ-sec funnel-metadynamics simulations, which efficiently enhance the sampling of bound and unbound states by using a funnel-shaped restraining potential to limit the exploration in the solvent. We reveal the sequence of events in the binding process from the capture of GABA from the solvent to its pinning between the charged residues Arg111 and Glu204 in the binding pocket. We characterize the associated free energy landscapes in the wild-type RDL receptor and in two mutant forms, where the key residues Arg111 and Glu204 are mutated to Ala. Experimentally these mutations produce nonfunctional channels, which is reflected in the reduced ligand binding affinities due to the loss of essential interactions. We also analyze the dynamical behavior of the crucial loop C, whose opening allows the access of GABA to the binding site and closure locks the ligand into the protein. The RDL receptor shares structural and functional features with other pLGICs; hence, our work outlines a valuable protocol to study the binding of ligands to pLGICs beyond conventional docking and molecular dynamics techniques.

Highlights

Pentameric ligand-gated ion channels are important neuroreceptors involved in fast synaptic communication and many neurological disorders.[1]
We monitor the conformational stability of the resistance to dieldrin (RDL) receptor models by calculating the root-mean-square deviation (RMSD) of the secondary structure backbone atoms with respect to the corresponding initial minimized structures
We have applied the enhanced sampling method funnelmetadynamics to investigate the binding mechanism of the neurotransmitter GABA to the wild-type and the most relevant mutant forms of the insect RDL receptor, a prototypical Pentameric ligand-gated ion channels (pLGICs). This method allows us to overcome the limitations of conventional docking and molecular dynamics (MD)-based techniques by restricting the exploration of the unbound region so to sample a large number of binding and unbinding events within an affordable computational time

Summary

Introduction

Pentameric ligand-gated ion channels (pLGICs) are important neuroreceptors involved in fast synaptic communication and many neurological disorders.[1] They are targets for drugs and, in invertebrates, for insecticides They are membrane proteins composed of five subunits arranged around an ion permeable channel with an extracellular domain (ECD), a transmembrane domain (TMD) spanning the cell membrane, and often an intracellular domain (ICD). Spectroscopic techniques and/or homology modeling provide the atomistic structure of these receptors’ ECD, which contains the relevant region for ligand binding This information has been used in docking and molecular dynamics (MD) calculations to study the binding modes of ligands to pLGICs. the approximate description of ligand/ protein interactions by docking algorithms and the limited time scale of MD simulations allow only a partial understanding of the binding process. A comprehensive elucidation of the activation mechanisms in pLGICs needs detailed information on the ligand binding steps and an accurate description of the free energy landscape, which remain elusive to standard simulation techniques and require the use of more sophisticated calculations.[2]

Methods

Results

Conclusion