Figure 1.
Some classical GABAA receptor ligands.
GABA is the endogenous GABAR agonist, muscimol a classical high-affinity agonist and THIP a muscimol analogue. Although not a GABAR ligand, glutamate is included to illustrate the resemblance to GABA. Diazepam (DZP) belongs to the benzodiazepine class of compounds, which are allosteric GABAA modulators. The DZP-NCS analogue attaches covalently to GABAARs and is included for validation purposes.
Figure 2.
Illustration of the GABAAR structural composition.
A) Top view showing the pentameric assembly of α1, β2 and γ2 subunits and the location of binding sites for GABA and BZDs; B) Side view illustrating the extracellular domain (ECD) where agonists and benzodiazepines bind and the transmembrane domain (TMD); C) Zooming in on a GABA binding site at the subunit interface between β2 and α1 subunits, loop regions A–F mentioned in the text are shown (A: yellow, B: orange, C: red, D: purple, E: blue and F: pink).
Figure 3.
Alignment of protein sequences from GluCl, GLIC, ELIC and the human α1, β2, and γ2 GABAAR subunits.
The GluCl sequence was used as template for homology modeling throughout the GABAAR subunits, and ELIC was included as a template in the regions marked with blue boxes. The secondary structure deduced from the X-ray structure of GluCl is shown above the alignment (red shapes denote α-helices, and green arrows denote β-strands), whereas historically assigned loop regions are indicated below the alignment. As in the GluCl structure the M3–M4 intracellular loop of GABAAR sequences was replaced by an AGT tri-peptide linker. Residues comprising the binding sites (within 8 Å of Glu in the GluCl structure and pointing towards the binding site) are colored pink (Glu and GABA binding site) and cyan (BZD binding site). Binding site residues conserved with respect to the templates are indicated as follows: + conserved in both GABA and BZD binding sites; * conserved in the GABA binding sites; . conserved in the BZD binding site. For details on calculation of binding site sequence identities see Figure S1.
Table 1.
Overview of mutations affecting the function of the GABAAR or the binding of orthosteric ligands.
Table 2.
Overview of mutations affecting the function of the GABAAR or the ability of BZD binding site ligands to bind to the BZD binding site.
Figure 5.
Agonist binding modes determined by induced fit docking.
A) GABA (green), B) THIP (pink) and C) muscimol (cyan) are shown in the orthosteric binding site at the interface between the β2 subunit (teal) and the α1 subunit (smudge).
Figure 4.
The figure shows residues that are conserved or homologous to GABAAR binding site residues from the GluCl X-ray structure (PBD ID 3RIF) as grey sticks and the bacterial Cys-Loop receptor homolog, ELIC (PDB ID 2VL0) as purple sticks. Glutamate as co-crystallized with GluCl is shown in yellow, where the structure corresponding to GABA is shown as sticks and the α-carboxylic acid removed prior to homology modeling is shown as lines. A) GABA binding site and B) BZD binding site.
Figure 6.
Conformational energy profile for dihedral drive of the amino-methyl side chain of muscimol.
B3LYP/6-31G** energies.
Figure 7.
Region suited for a tightly bound water molecule identified in agonist site.
A GRID calculation at the agonist binding site, using the water probe, identified two regions of strong binding interaction energy (−11 kcal/mol). One region is overlapping with the acidic moiety of agonists and the other region is situated next to the backbone of β2S156 and β2Y157 (grey mesh). The calculation was performed in absence of agonist in the binding site. In the picture, the site has been optimized for muscimol as described in the methods section. A) When a water molecule is placed between muscimol and the B-loop backbone, perfect hydrogen bonding distances are obtained, resulting in optimal interactions between the high affinity ligand muscimol and the GABA receptor. B) When also GABA is included in the site, it is obvious that the water molecule would make up for the backbone interaction that GABA is predicted to make.
Figure 8.
A) The assumed biologically active binding mode of DZP (gray) at the interface between the α1 (smudge) and γ2 (firebrick) subunits. In this conformation the C-3 points upwards and the pending phenyl substituent is directed inwards. B) Covalently attached DZP-NCS (cyan) overlaid with DZP (gray). Only moderate differences between the docked and the covalently attached ligands exist.