Insights into the binding of GABA to the insect RDL receptor from atomistic simulations: a comparison of models

Abstract

The resistance to dieldrin (RDL) receptor is an insect pentameric ligand-gated ion channel (pLGIC). It is activated by the neurotransmitter γ-aminobutyric acid (GABA) binding to its extracellular domain; hence elucidating the atomistic details of this interaction is important for understanding how the RDL receptor functions. As no high resolution structures are currently available, we built homology models of the extracellular domain of the RDL receptor using different templates, including the widely used acetylcholine binding protein and two pLGICs, the Erwinia Chrysanthemi ligand-gated ion channel (ELIC) and the more recently resolved GluCl. We then docked GABA into the selected three dimensional structures, which we used as starting points for classical molecular dynamics simulations. This allowed us to analyze in detail the behavior of GABA in the binding sites, including the hydrogen bond and cation-π interaction networks it formed, the conformers it visited and the possible role of water molecules in mediating the interactions; we also estimated the binding free energies. The models were all stable and showed common features, including interactions consistent with experimental data and similar to other pLGICs; differences could be attributed to the quality of the models, which increases with increasing sequence identity, and the use of a pLGIC template. We supplemented the molecular dynamics information with metadynamics, a rare event method, by exploring the free energy landscape of GABA binding to the RDL receptor. Overall, we show that the GluCl template provided the best models. GABA forming direct salt-bridges with Arg211 and Glu204, and cation-π interactions with an aromatic cage including Tyr109, Phe206 and Tyr254, represents a favorable binding arrangement, and the interaction with Glu204 can also be mediated by a water molecule.

Fig. 1

Sequence alignments of RDL with AChBP (~20 % sequence identity), ELIC (~24 %) and GluCl (~39 % for GluCl1 and ~38 % for GluCl2). The residues studied by mutagenesis experiments [17, 18] are highlighted (principal subunit: Phe146, Glu204, Phe206 and Tyr254; complementary subunit: Tyr109; Arg111 and Ser176)

Fig. 2

The selected homology models for the pentameric extracellular domain of the RDL receptor, after structure optimization: a RDL-AChBP; b RDL-ELIC; c RDL-GluCl1; d RDL-GluCl2. The seven residues Phe146, Glu204, Phe206 and Tyr254 in the principal subunit and Tyr109, Arg111 and Ser176 in the complementary subunit are explicitly shown. The displacement of the protein backbone from that of the RDL-GluCl2 model is highlighted

Fig. 3

The GABA binding sites consisting of residues Phe146, Glu204, Phe206, Tyr254, Tyr109, Arg111 and Ser176, in the optimized homology models before GABA docking: a RDL-AChBP; b RDL-ELIC; c RDL-GluCl1; d RDL-GluCl2. The displacement of each atom from its position in the RDL-GluCl2 model is highlighted

Fig. 4

Example of GABA docked in the extracellular domain of the RDL receptor (RDL-GluCl1 model), with residues in the binding pocket. Labels to selected GABA atoms are shown. The dashed lines represent the collective variables used in the metadynamics simulation

Fig. 5

Root mean square displacements of the backbone atoms during the molecular dynamics simulations, including equilibration with the gradual release of restraints, for the RDL receptor models

Fig. 6

Time evolution (top) of the dihedral angles ψ and ϑ which characterize the GABA conformers, and conformer relative occupancy (bottom) during molecular dynamics simulations in a water, b RDL-AChBP-5, c RDL-ELIC-5, d RDL-GluCl1-5 and e RDL-GluCl2-5. The time evolutions of the five ligands are superimposed and the occupancies are averaged over the five binding sites

Fig. 7

Molecular dynamics snapshots of GABA in the binding site of RDL-GluCl1 (left) and RDL-GluCl2 (right). Hydrogen bond interactions between GABA and the RDL receptor models are indicated with dashed lines

Fig. 8

Left: Free energy map of GABA in the RDL-GluCl1 model as a function of the distance of the GABA amine from Glu204 side chain (CV_Glu) and of the GABA carboxylate from the Arg111 side chain (CV_Arg). Right: Binding arrangements corresponding to the free energy minima