Homodimerization enhances both sensitivity and dynamic range of the ligand-binding domain of type 1 metabotropic glutamate receptor

Abstract

Cooperativity in ligand binding is a key emergent property of protein oligomers. Positive cooperativity (higher affinity for subsequent binding events than for initial binding) is frequent. However, the symmetrically homodimeric ligand-binding domain (LBD) of metabotropic glutamate receptor type 1 exhibits negative cooperativity. To investigate its origin and functional significance, we measured the response to glutamate in vitro of wild-type and C140S LBD as a function of the extent of dimerization. Our results indicate that homodimerization enhances the affinity of the first, but not the second, binding site, relative to the monomer, giving the dimeric receptor both greater sensitivity and a broader dynamic range.

Keywords: G-protein coupled receptor; cooperativity; ligand binding; light scattering; venus flytrap fold.

Figure 1

(A) Crystal structure of the asymmetric mGluR1 LBD dimer (PDB ID 1EWK). (B) Schematic representation of activation of mGluR1 LBD in the dimeric and monomeric form. D – dimer; M – monomer; the bi-lobed VFT domains are shown in gray.

Figure 2

(A) SDS-PAGE in the presence (+) or absence (−) of β-mercaptoethanol (βME) shows the switch from dimer to monomer upon mutation. (B, C) Representative samples of purified WT and C140S LBD characterized by size-exclusion chromatography. The first peak in (C) is attributable to aggregated C140S LBD. Only the peak 1 ml fractions were used for further analysis. Reruns of the 12–13 ml C140S fraction on the SEC column did not show an aggregate peak over 1 week of storage.

Figure 3

Weight-average molecular weight of C140S samples (determined from light scattering) as a function of total subunit concentration (determined from refractive index). (A) Representative size-exclusion traces with MALLS measurements for the top 0.3 ml of the absorbance peak overlaid. (B) Fitting the SEC/MALLS data for three separate batches of C140S (indicated by distinct symbols) without Glu (solid line fit) and one batch with saturating Glu (dashed line fit). When saturating glutamate is present, the equilibrium shifts toward tighter dimerization, indicating that ligand binding does not disrupt the dimer, but stabilizes it. Standard errors were determined according to [53].

Figure 4

(A) Representative traces of intrinsic fluorescence change in the WT mGluR1 LBD upon addition of saturating [glutamate]. (B) Titrations of 8 nM WT LBD (black), 70 nM C140S LBD (blue), and 8 nM C140S LBD (red) with glutamate. The ordinate represents the percent enhancement of intrinsic tryptophan fluorescence upon application of a given concentration of glutamate. Each point is the average of 2–3 independent experiments; standard error of the mean is ~1 (% Glu response) for each point. All data are fitted to the Adair model (solid lines).