Mechanisms of Glutamate Capture by a Clamshell Binding Domain

Abstract

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels responsible for the majority of excitatory synaptic transmission in the brain. Glutamate binding into the clamshell-like ligand-binding domains (LBDs) triggers a conformational change that opens the channel pore, resulting in receptor activation. The stable glutamate-bound conformation of the LBD has been well-studied structurally, but the dynamic process of ligand binding is poorly understood. To elucidate this process, we performed microsecond-timescale molecular dynamics (MD) simulations of glutamate binding to an AMPA receptor LBD together with electrophysiological studies. Potential of mean force (PMF) calculations identify several metastable glutamate interaction regions outside of the binding pocket. Glutamate is observed to bind in two distinct conformations, one of which corresponds to the crystallographically-observed conformation. We evaluated the kinetics of various dynamical processes using a Markov state model analysis. Using the simulations as a guide, a battery of mutants was designed to target the metastable regions, replacing charged residues with either neutral ones or those of opposite charge. Some of these mutants slowed activation and deactivation of the receptor, consistent with slowed association and dissociation of glutamate, whereas off pathway mutations had little effect. Our results suggest that glutamate binding in iGluRs occurs through multiple, independent pathways, and that the ligand interacts strongly with several key residues along these pathways.