Ionotropic glutamate receptors (iGluRs) are membrane proteins which act as molecular pores and mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. The 7 gene families of ionotropic glutamate receptors (iGluRs) in humans encode 18 subunits which assemble to form 3 major functional families named after the ligands which were first used to identify iGluR subtypes in the late 1970s: AMPA, kainate and NMDA. Because of their essential role in normal brain function and development, and increasing evidence that dysfunction of iGluR activity mediates multiple neurological and psychiatric diseases, as well as damage during stroke, a substantial effort in the Laboratory of Cellular and Molecular Neurophysiology is directed towards analysis of iGluR function at the molecular level. Atomic resolution structures solved by protein crystallization and X-ray diffraction provide a framework in which to design electrophysiological and biochemical experiments to define the mechanisms underlying ligand recognition, the gating of ion channel activity, and the action of allosteric modulators. This information will allow the development of subtype selective antagonists and allosteric modulators with novel therapeutic applications and reveal the inner workings of a complicated protein machine which plays a key role in brain function. Crystallographic and functional analysis of an allosteric binding site for sodium Kainate subtype glutamate receptors are strongly modulated by monovalent anions and cations. In the absence of either chloride or sodium the receptors become non functional. A combined experimental approach using crystallography, patch clamp recording, and all atom molecular dynamics simulations was used to identify the binding site for sodium, and the mechanism by which sodium modulates kainate receptor activity. Structures were solved for the GluR5 ligand binding domain dimer complex with lithium, sodium, potassium, rubidium, cesium and ammonium ions in the cation binding site. There are two sodium binding sites in a dimer assembly, one per subunit, and these flank the previously identified anion binding site which lies on the molecular two-fold axis of symmetry. Sodium acts by stabilizing the dimer assembly in its active conformation required for ion channel gating, and in the absence of sodium the receptors desensitize much faster. Sodium selectivity is conferred by a high electric field strength in the cation binding site, but larger cations can bind with lower affinity. Functional studies show that the cation binding site is allosterically coupled to the anion binding site. All atom MD simulations and free energy calculations reveal that the binding of chloride is favored by 3-5 kcal per mole when the cation binding site is occupied by sodium. Mutational analysis and molecular modeling revealed that it is possible to convert the sodium binding site to one which has micromolar affinity for the divalent cations calcium and magnesium. This was achieved by substituting an aspartate residue for a hydrophobic amino acid which caps the sodium binding site in kainate receptors. AMPA receptors, which are insensitive to allosteric modulation by either sodium or calcium harbor a lysine at this site. Amino acid sequence analysis indicates that the divergence between iGluRs with and without allosteric binding sites for sodium arose early in evolution. We were unable to crystallize a kainate receptor with the aspartate mutation, and so made molecular models of the binding site. The model reveals a unique geometry, with three closely apposed carboxylate groups, together with two backbone carbonyl oxygen atoms, that provides ligands for binding calcium similar to those found in the protein databank for a diverse range of proteins with calcium binding sites. Direct measurements of the effects of allosteric ions on dimer assembly by kainate subtype iGluRs is not possible using electrophysiological techniques. In order to obtain proof that allosteric ions regulate dimer formation, a series of GluR6 dimer interface mutations, remote from the ion binding sites, was developed with the goal of developing a preparation amenable to analysis by analytical ultracentrifugation (AUC). The mutants were designed on the basis of crystal structures for wild type GluR5 dimers, using electrophysiological analysis to search for a phenotype with slowed kinetics of desensitization. The isolated ligand binding domains of the same mutants were prepared and their affinity for dimer formation measured both by sedimentation velocity analysis over a range of protein concentrations, and by sedimentation equilibrium. Structural studies on the amino terminal domain of iGluRs Glutamate receptor ion channels are multidomain membrane proteins which assemble of tetramers of molecular weight approximately 440 kD. Numerous crystal structures have been solved for the ligand binding domains which have a molecular weight of approximately 30 kD per subunit, approximately of the mass of an intact receptor. Extensive trials with bacterial expression systems, which with one exception, have been used for all published ligand binding domain structures, failed to produce monodisperse soluble protein for other iGluR domains. The amino terminal domain (ATD) is an important structural target because it controls subtype selective assembly in native iGluRs, limiting assembly to members of the same functional family. Protein expression at levels sufficient for structural biology in mammalian cells is much more difficult than expression in E.coli but has the advantages that multiple check points select for correctly folded proteins, and add sugars and other post translational modifications required for normal function. Although a variety of cell biological and biochemical techniques are required to subsequently trim the sugar chains, in order to obtain proteins which crystallize and diffract to high resolution, and the yields are lower than for prokaryotic expression, currently this is the only approach likely to succeed for studies of the ATD. In ongoing work the ATDs from several iGluR subtypes have been screened for expression in mammalian cells. Crystallization trials have been performed using a nano liter pipetting robot, and for one diffraction data to a resolution of 2.65 for a complete data set was obtained at APS. Structure solution and refinement is complete. Structural analysis of NR3 ligand binding selectivity NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well characterized members of the NMDA receptor gene family which play key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations we investigated mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. Replica exchange MD simulations of 650 ns duration revealed numerous interdomain contacts which stabilize the agonist bound closed cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits. Mutation of these sites destabilized ligand binding measured by titration assays using quenching of endogenous tryptophan fluorescence.
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