Abstract
Protein dimerization provides a mechanism for the modulation of cellular signaling events. In α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, the rapidly desensitizing, activated state has been correlated with a weakly dimeric, glutamate-binding domain conformation. Allosteric modulators can form bridging interactions that stabilize the dimer interface. While most modulators can only bind to one position with a one modulator per dimer ratio, some thiazide-based modulators can bind to the interface in two symmetrical positions with a two modulator per dimer ratio. Based on small-angle X-ray scattering (SAXS) experiments, dimerization curves for the isolated glutamate-binding domain show that a second modulator binding site produces both an increase in positive cooperativity and a decrease in the EC50 for dimerization. Four body binding equilibrium models that incorporate a second dimer-stabilizing ligand were developed to fit the experimental data. The work illustrates why stoichiometry should be an important consideration during the rational design of dimerizing modulators.