Glutamate Receptor Structural Biology

Mark L. Mayer, PhD, Head, Section on Neurophysiology and Biophysics
Charu Chaudhry, PhD, Postdoctoral Fellow
Janesh Kumar, PhD, Postdoctoral Fellow
Andrew Plested, PhD, Postdoctoral Fellow
Yongneng Yao, PhD, Postdoctoral Fellow
Carla Glasser, BS, Technical Specialist

Ionotropic glutamate receptors (iGluR) are membrane proteins that act as molecular pores and mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. The seven gene families iGluRs in humans encode 18 subunits, which assemble to form three major functional families named after the ligands that were first used to identify iGluR subtypes in the late 1970s: AMPA, kainite, and NMDA. Given iGluRs’ essential role in normal brain function and development and mounting evidence that dysfunction of iGluR activity mediates several neurological and psychiatric diseases and damage during stroke, we devote substantial effort to analyzing iGluR function at the molecular level. Atomic resolution structures solved by protein crystallization and X-ray diffraction provide a framework for designing electrophysiological and biochemical experiments aimed at defining the mechanisms underlying ligand recognition, the gating of ion channel activity, and the action of allosteric modulators. Information derived from these experiments will permit the development of subtype-selective antagonists and allosteric modulators with novel therapeutic applications and reveal the inner workings of a complicated protein machine that plays a key role in brain function.

Crystallographic and functional analysis of an allosteric binding site for sodium

Plested, Mayer; in collaboration with Biggin

Kainate-subtype glutamate receptors are strongly modulated by monovalent anions and cations. In the absence of either chloride or sodium, the receptors become nonfunctional. We used a combined experimental approach based on crystallography, patch-clamp recording, and all-atom molecular dynamics (MD) simulations to identify the binding site for sodium and the mechanism by which sodium modulates kainate receptor activity. We solved structures for the GluR5 ligand–binding domain dimer complex with lithium, sodium, potassium, rubidium, cesium, and ammonium ions in the cation-binding site. We observed two sodium binding sites in a dimer assembly (one per subunit); they flank the previously identified anion-binding site that lies on the molecular two-fold axis of symmetry. Sodium stabilizes the dimer assembly in its active conformation, which is required for ion channel gating; 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 to 5 kcal per mole when the cation-binding site is occupied by sodium. Mutational analysis and molecular modeling demonstrated that it is possible to convert the sodium-binding site to a site with micromolar affinity for the divalent cations calcium and magnesium, namely by substituting an aspartate residue for a hydrophobic amino acid that 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 indicated 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 therefore 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 provide ligands for binding to calcium similar to those found in the protein data bank for a diverse range of proteins with calcium binding sites.

Plested AJR, Mayer ML. Engineering a high-affinity allosteric binding site for divalent cations in kainate receptors. Neuropharmacology 2008; [E-pub ahead of print].
Plested AJR, Mayer ML. Structure and mechanism of kainate receptor modulation by anions. Neuron 2007;53:829-841.
Plested AJR, Vijayan R, Biggin P, Mayer ML. Molecular basis of kainate receptor modulation by sodium. Neuron 2008;58:720-735.

Molecular biophysical studies on kainate receptor dimer assembly

Chaudhry, Mayer; in collaboration with Rosenmund, Schuck

Direct measurement of the effects of allosteric ions on dimer assembly by kainate subtype iGluRs is not possible with electrophysiological techniques. To obtain proof that allosteric ions regulate dimer formation, we developed a series of GluR6 dimer interface mutations remote from the ion-binding sites and set out to develop a preparation amenable to analysis by analytical ultracentrifugation (AUC). We designed the mutants on the basis of crystal structures for wild-type GluR5 dimers, using electrophysiological analysis to search for a phenotype with slowed kinetics of desensitization. We prepared the isolated ligand-binding domains of the same mutants and measured their affinity for dimer formation by sedimentation velocity analysis over a range of protein concentrations and by sedimentation equilibrium. The most stable mutants increased dimer stability by at least 3 kcal per mole, with excellent agreement between the Kd for dimer assembly and the rate of desensitization measured in functional experiments. To explore the molecular basis for control of dimer assembly, we crystallized the wt GluR6 dimer together with a series of six mutants at resolutions of 1.2 to 1.5 Å. Analysis of these structures and of the effects of allosteric ions on dimer assembly measured by AUC is in progress.

Weston MC, Schuck P, Ghosal A, Rosenmund C. Conformational restriction blocks glutamate receptor desensitization. Nat Struct Mol Biol 2006;13:1120-1127.

Structural studies on the amino-terminal domain of iGluRs

Kumar, Mayer

Glutamate receptor ion channels are multidomain membrane proteins that assemble from tetramers of 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 one-quarter 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 several 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 that crystallize and diffract to high resolution, and the yields are lower than for prokaryotic expression, mammalian cell culture is the only current approach likely to succeed for studies of the ATD. In ongoing work, we have screened the ATDs from several iGluR subtypes for expression in mammalian cells. We have performed crystallization trials using a nanoliter pipetting robot and have obtained, for one diffraction, data to a resolution of 2.65 Å for a complete data set at APS. Structure solution and refinement is in progress.

Structural analysis of NR3 ligand binding selectivity

Yao, Mayer; in collaboration with Schulten

NR3-subtype glutamate receptors have a unique developmental expression profile but are the least characterized members of the NMDA receptor gene family; those members 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 of NR3A and NR3B to 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 to that found in the corresponding NR1 complexes but have a unique loop 1 structure distinct from those 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 in NR1 and fail to undergo a conformational rearrangement observed in NR1 upon binding to the partial agonist ACPC. Replica exchange MD simulations of 650 ns duration revealed numerous interdomain contacts that stabilize the agonist-bound closed-cleft conformation as well as a novel twisting motion for the loop 1 helix that is unique in NR3 subunits. Mutation of these sites destabilized ligand binding as measured by titration assays that use quenching of endogenous tryptophan fluorescence.

Yao Y, Harrison CB, Freddolino PL, Schulten K, Mayer ML. Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors. EMBO J 2008;27:2158-2170.

Collaborators

Philip Biggin, PhD, University of Oxford, Oxford, UK
David Jane, PhD, University of Bristol, Bristol, UK
Christian Rosenmund, PhD, Baylor College of Medicine, Houston, TX
Peter Schuck, PhD, Protein Biophysics Resource, DBEPS, NIH, Bethesda, MD
Klaus Schulten, PhD, University of Illinois at Urbana-Champaign, Urbana, IL

For further information, contact mlm@helix.nih.gov or visit http://snb.nichd.nih.gov.