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The GABAC receptor ligand binding site: a modelling, docking and mutagenesis study
The GABAC receptor is a member of the Cys-loop family of ligand-gated ion channels, which includes the nicotinic acetylcholine (nACh), GABAA, glycine and 5-HT3 receptors (Chebib, 2004). There have been no high resolution images produced of the binding site of these proteins, yet the AChBP (Brejc et al., 2001), a protein homologous to the extracellular domain of the nACh receptor, has been crystallised and provides a candidate protein for homology modelling of this region. The aim of this work was to create a model of the binding site of the GABAC receptor, dock GABA, and then experimentally verify the results. A sequence alignment was made of the ρ1 subunit sequence with an AChBP monomer using FUGUE (Shi et al., 2001). From this alignment a 3-D model was built using MODELLER (Sali et al., 1993), based on the AChBP crystal structure. The pentamer was generated by superimposing the model onto each protomer of AChBP. The pentamer was then energy minimised using SYBYL in the AMBER force field (Weiner et al., 1984). The resultant structure was composed predominantly of β-sheets, with a cavity containing residues identified as important for GABA binding. GABA was docked into this cavity using FlexX (Accelerys), which revealed three potential orientations, based on those amino acids which were within 5Å of the ligand. In total 17 amino acids were implicated in such close proximity, although only 3 residues were common to all orientations: S168, S243 and Y198. The specific interactions at Y198 have been probed using unnatural amino acid mutagenesis, which involved the introduction of a number of Phe derivatives at this position, (Lummis et al., 2005). These receptors were expressed in Xenopus oocytes and tested for responses to GABA using the two-electrode voltage clamp method. The data obtained demonstrated a strong correlation between cation-π binding ability of the incorporated residue and the log(EC 50) for GABA. This suggested a cation-π interaction between the aromatic of Y198 and the positive amine of GABA. Only one orientation of GABA satisfied this cation-π interaction, with the amine group also stabilised through H-bonds to S168 and L166. In this orientation the negative carboxylate of GABA interacted with the R104, making two H-bonds with this residue. In conclusion, we have identified an orientation of GABA in its binding site which satisfies the zwitterionic properties of the ligand, and is supported by experimental data. The development of such ligand binding models in the GABAC receptor could be utilised in the development of drugs that target this receptor, and may also serve as a reference for the related GABAA receptor.
Brejc, K. et al. (2001) Nature 411, 269-87. |
