Figure 5: Thermodynamic dissection of the subunit specificity of gephyrin.

(a) Different contributions of the binding enthalpy and entropy to the overall free energy. Residues conserved among the GlyR β and GABA_AR α3 subunits are shown in magenta, GlyR β residues in blue and GABA_AR α3 residues in black. The GlyR β peptide displayed a 23-fold higher affinity than the corresponding GABA_AR peptide. Subunit specific residues were exchanged between both receptor subunits. Note that the gephyrin affinity can be maximized by combining hydrophobic residues of GlyRs and GABA_ARs (GlyR S403T and GABA_AR P405Y). Remarkably, full GlyR-like binding affinity can be reconstituted for the GABA_AR fragment by only two mutations, N369S and T374L. Enthalpy, entropy and free energy bar graphs are averages (with their standard deviations indicated by the error bars) of at least three ITC measurements. (b) Differential binding of GABA_ARs and GlyRs to an overlapping gephyrin-binding site. Structures of the GlyR and GABA_AR peptides in stick representation colour-coded according to the relative binding contribution (high (>1 kcal mol⁻¹) in red, low (<1 kcal mol⁻¹) in orange and none (~0 kcal mol⁻¹) in blue) with the gephyrin surface shown in grey. The corresponding sequences of the full-length intracellular loops of GABA_AR α3 and GlyR β are shown below using the same colour code. Note that different ligand residue positions mediate the critical gephyrin interactions.