GABA Binding to an Insect GABA Receptor: A Molecular Dynamics and Mutagenesis Study

Abstract

RDL receptors are GABA-activated inhibitory Cys-loop receptors found throughout the insect CNS. They are a key target for insecticides. Here, we characterize the GABA binding site in RDL receptors using computational and electrophysiological techniques. A homology model of the extracellular domain of RDL was generated and GABA docked into the binding site. Molecular dynamics simulations predicted critical GABA binding interactions with aromatic residues F206, Y254, and Y109 and hydrophilic residues E204, S176, R111, R166, S176, and T251. These residues were mutated, expressed in Xenopus oocytes, and their functions assessed using electrophysiology. The data support the binding mechanism provided by the simulations, which predict that GABA forms many interactions with binding site residues, the most significant of which are cation-π interactions with F206 and Y254, H-bonds with E204, S205, R111, S176, T251, and ionic interactions with R111 and E204. These findings clarify the roles of a range of residues in binding GABA in the RDL receptor, and also show that molecular dynamics simulations are a useful tool to identify specific interactions in Cys-loop receptors.

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

“…The selected alignments are shown in Fig. 1, where the residues studied by mutagenesis experiments [17, 18] are highlighted. As particular attention was given to reproduce realistic binding sites, the alignment of other parts of the protein, such as the variable region between the β8 and β9 strands, may be less accurate.

Fig.

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“…The residues studied by mutagenesis experiments [17, 18] are highlighted (principal subunit: Phe146, Glu204, Phe206 and Tyr254; complementary subunit: Tyr109; Arg111 and Ser176)…”

“…GABA was docked in RDL-GluCl2 within a 10 Å radius from Phe206 [18], with the docking software GOLD 4.0 [44]. Optimal activation of pLGICs requires the binding of at least two agonists likely in non-consecutive interfaces, but for the investigation of the binding network with MD simulations the interfaces can be considered independent; in fact some MD studies simulate only two subunits rather than the whole pentamer [45].…”

Synthesis of 1,3-di- and 1,3,4-trisubstituted 1,6-dihydro-6-iminopyridazines as competitive antagonists of insect GABA receptors

“…Both experimental evidence and molecular dynamics simulation indicated that the amino acid residue corresponding to Arg109 plays a critical role in the interaction with GABA in Drosophila Rdl GABAR. 38) The two aromatic amino acids, Phe204 in loop B and Tyr252 in loop C, surround the protonated amino group of GABA and may produce cation-π interactions, as proposed for Drosophila Rdl GABARs. [38][39][40] The bound GABA is in an extended conformation that is similar to that of the Drosophila Rdl GABAR and GABA C R but not to that of the GABA A R. 38,40) Several amino acids located in loops D, E, and F of the α1 and ρ1 subunits and those in loop A and C of the β2 and ρ1 subunits in GABA A Rs and GABA C Rs have been identified as gabazineinteracting residues by site-directed mutagenesis and functional analysis of the mutants.…”

“…38) The two aromatic amino acids, Phe204 in loop B and Tyr252 in loop C, surround the protonated amino group of GABA and may produce cation-π interactions, as proposed for Drosophila Rdl GABARs. [38][39][40] The bound GABA is in an extended conformation that is similar to that of the Drosophila Rdl GABAR and GABA C R but not to that of the GABA A R. 38,40) Several amino acids located in loops D, E, and F of the α1 and ρ1 subunits and those in loop A and C of the β2 and ρ1 subunits in GABA A Rs and GABA C Rs have been identified as gabazineinteracting residues by site-directed mutagenesis and functional analysis of the mutants. [41][42][43][44][45][46][47] The docking studies of 7e using an HF GABAR homology model showed that the side chain carboxylate of Glu202 in loop B functions as an acceptor of the protonated imino hydrogen and that the guanidino group of Arg109 in loop D serves as a hydrogen donor for the lone pair of electrons of the nitrile nitrogen (Fig.…”