Agonist-induced isomerization in a glutamate receptor ligand-binding domain - A kinetic and mutagenetic analysis

Agonist binding to glutamate receptor ion channels occurs within an extrace llular domain (S1S2) that retains ligand affinity when expressed separately . S1S2 is homologous to periplasmic binding proteins, and it has been propo sed that a Venus flytrap-style cleft closure triggers opening of glutamate receptor ion channels. Here we compare the kinetics of S1S2-agonist binding to those of the periplasmic binding proteins and show that the reaction in volves an initial rapid association, followed by slower conformational chan ges that stabilize the complex: "docking" followed by "locking." The motion detected here reflects the mechanism by which the energy of glutamate bind ing is converted into protein conformational changes within S1S2 alone. In the intact channel, these load-free conformational changes are harnessed an d possibly modified as the agonist binding reaction is used to drive channe l opening and subsequent desensitization. Using mutagenesis, key residues i n each step were identified, and their roles were interpreted in light of a published S1S2 crystal structure. In contrast to the Venus flytrap proposa l, which focuses on motion between the two lobes as the readout for agonist binding, we argue that smaller, localized conformational rearrangements al low agonists to bridge the cleft, consistent with published hydrodynamic me asurements.