Hydrophobic Side Chain Dynamics of a Glutamate Receptor Ligand Binding Domain

Alexander S Maltsev,Robert E Oswald

doi:10.1074/jbc.m109.088641

Abstract

Ionotropic glutamate receptors are ligand-gated ion channels that mediate much of the fast excitatory neurotransmission in the central nervous system. The extracellular ligand binding core (S1S2) of the GluR2 subtype of ionotropic glutamate receptors can be produced as a soluble protein with properties essentially identical to the corresponding domain in the intact, membrane-bound protein. Using a variety of biophysical techniques, much has been learned about the structure and dynamics of S1S2 and the relationship between its ligand-induced conformational changes and the function of the receptor. It is clear that dynamic processes are essential to the function of ionotropic glutamate receptors. We have isotopically labeled side chain methyls of GluR2 S1S2 and used NMR spectroscopy to study their dynamics on the ps-ns and mus-ms time scales. Increased disorder is seen in regions that are part of the key dimer interface in the intact protein. When glutamate is bound, the degree of ps-ns motion is less than that observed with other ligands, suggesting that the physiological agonist binds to a preformed binding site. At the slower time scales, the degree of S1S2 flexibility induced by ligand binding is greatest for willardiine partial agonists, least for antagonists, and intermediate for full agonists. Notable differences among bound ligands are in the region of the protein that forms a hinge between two lobes that close upon agonist binding, and along the beta-sheet in Lobe 2. These motions provide clues as to the functional properties of partial agonists and to the conformational changes associated with lobe closure and channel activation.

Highlights

Brain damage and neurodegenerative disorders such as Huntington chorea, Parkinson, and Alzheimer diseases. iGluRs are membrane-bound receptor ion channels composed of four subunits surrounding a central ion channel in which each subunit contributes to pore formation
We report here on the motions of isoleucine, valine, and leucine methyl groups on the ps-ns and ␮s-ms time scales studied using NMR spectroscopy in combination with specific 13C and 1H labeling of the GluR2 S1S2 domain
Side Chain Dynamics of GluR2 faster time scale, binding of glutamate was found to induce a higher average order parameter of the methyl groups compared with all other ligands, possibly reflecting the fact that it is the natural ligand for this receptor and produces the least perturbation of the hydrophobic packing

Summary

Introduction

Brain damage and neurodegenerative disorders such as Huntington chorea, Parkinson, and Alzheimer diseases. iGluRs are membrane-bound receptor ion channels composed of four subunits surrounding a central ion channel in which each subunit contributes to pore formation. The large scale mechanisms (e.g. lobe closure is associated with channel activation) are relatively clear from the crystal structures, the kinetic and thermodynamic details of activation and desensitization require analysis by methods that can accurately detect dynamic processes (NMR, IR, etc.). This has led to additional insights into the function of these important neurotransmitter receptors. These motions as well as those near the hinge axis may provide additional details concerning processes associated with channel activation

Results

Discussion

Conclusion