Conformational Dynamics of Ligand Binding Domains of GluR2

National Heart, Lung, And Blood Institute

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Abstract

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory signaling in the central nervous system. Upon ligand binding to the extracellular domain of iGluRs local conformational changes ensue and this motion is translated to the transmembrane domain inducing channel opening. We have used an isolated ligand binding domain (LBD), GluR2-S1S2J (GluR2), as a model system to study the protein-ligand complex. Using time-resolved fluorescence and anisotropy measurements, we characterized the excited state properties and local mobility of Trp residues in the isolated LBD, GluR2. Specifically, we determined that the widely used and structurally characterized antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX) acts as an efficient fluorescence energy transfer (FET) acceptor for Trp. Consistent with crystallographic data, our results indicate that the four native tryptophans are within Forsters radius (33 angstroms ) of the bound ligand. Additionally, we demonstrate the broader value of this technique by identifying an original FET ligand, 3-nitrotyrosine (3NY) for GluR2 (24 angstroms , apparent dissociation constant, Kd of approximately 170 micromolar). Estimated average donor-acceptor (Trp-to-ligand) distances extracted from tryptophan excited-state decays are similar for both ligands (24 angstroms) suggesting that 3NY binds in the structurally characterized ligand-binding cleft. Interestingly, we observe multiple rate components for 3NY suggestive of ligand-protein complex structural heterogeneity. However, due to the presence of multiple Trp donors we are only able to estimate average DNQX/3NY distances to the Trp residues. While the distance extracted (24 angstroms) from our analysis on DNQX-Trp GluR2 are on the order of the crystallographically determined distances (12-20 angstroms), they are somewhat longer. This may be due to the current limit of resolvable rates (tau of 0.5 ns) which would correspond to donor-acceptor distances shorter than those estimated for Trp-DNQX or it is possible that it is attributable to unfavorable orientations of the Trp and DNQX transition dipoles in the folded protein. Alternatively, our longer distances may simply reflect dynamical properties of the protein in solution. While DNQX is a well-known complex, its spectroscopic properties and potential application as a sensitive reporter for ligand binding to GluR2 have been overlooked previously. We have described a DNQX competition assay that represents a significant improvement over the fluorescence assays currently in use in that this is a turn-on sensor, eliminating false positive observations inherent to quenching events resulting from photobleaching. The use of FET competitor ligands and intrinsic Trp fluorescence should allow for the future identification of novel ligands for not only for GluR2 but also for other proteins when DNQX or structural analogs (i.e. CNQX and other nitrated aromatic compounds) are employed.