GLUTAMATE RECEPTOR STRUCTURAL BIOLOGY
, Head, Section on Neurophysiology and Biophysics
Yongneng Yao, PhD, Postdoctoral Fellow
Andrew Plested, PhD, Visiting Fellow
Carla Glasser, BS, Technical Specialist
Gregory Alushin, BS, Postbaccalaureate Fellow
Katelyn Lyons, BS, Postbaccalaureate Fellow
Ionotropic glutamate receptors (iGluRs) are membrane proteins that act as molecular pores and mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. In humans, the seven gene families of iGluRs encode 18 subunits that assemble to form three major functional families named after the ligands that, in the late 1970s, were first used to identify iGluR subtypes: AMPA, kainate, and NMDA. Given iGluRs’ essential role in normal brain function and development and increasing evidence suggesting that dysfunction of iGluR activity mediates several neurological and psychiatric diseases as well as damage during stroke, we direct substantial effort to analyzing GluR 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 in order to define the allosteric mechanisms underlying ligand recognition and the gating of ion channel activity. The resultant 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 that plays a major role in brain function.
Crystallographic and functional analysis of glutamate receptor ligand complexes
We solved high-resolution crystal structures for four GluR5 subtype–selective antagonist complexes; three antagonists are derivatives of the willardiine-based compounds for which we solved the first GluR5 antagonist complex in 2006, and one is with a decahydroisoquinoline, which has potential as an analgesic and a drug to reduce migraine. The structures reinforce the idea that, even when bound with competitive antagonists, glutamate receptors can sample a range of conformational space, similar in principle to the variation in domain closure observed for partial agonists, but distinct in that the difference in domain closure for individual antagonists does not cross the threshold necessary to trigger ion channel gating. One of the structures revealed a novel protein-ligand interaction, named a halogen bond, formed by a contact between a carboxylate side chain and a ligand bromine atom.
Dolman NP, More JCA, Alt A, Knauss JL, Pentikäinen OT, Glasser CR, Bleakman D, Mayer ML, Collingridge GL, Jane DE. Synthesis and pharmacological characterization of N3-substituted willardiine derivatives: role of the substituent at the 5-position of the uracil ring in the development of highly potent and selective GLUK5 kainate receptor antagonists. J Med Chem 2007;50:1558-70.
Mayer ML. Glutamate receptors at atomic resolution. Nature 2006;440:456-62.
Crystallographic and functional analysis of allosteric ion binding sites
Kainate subtype glutamate receptors are strongly modulated by monovalent anions and cations and, in the absence of either chloride or sodium, become nonfunctional. We used a combined experimental approach that involved crystallography and patch clamp recording to identify the binding site for anions. The chloride ion binds in the dimer interface between two subunits and acts as electrostatic glue, which helps stabilize dimer assemblies in their active conformation. In the absence of chloride, the dimers dissociate and the receptor desensitizes. Mutations that disrupt chloride binding have the same effect in functional experiments. Our results reveal that, instead of acting in a modulatory, allosteric manner, anions are essential structural components of the receptor in its active conformation. In ongoing work using the same approach, we are working to solve the structure of the cation binding site.
Mayer ML, Ghoshal A, Dolman NP, Jane DE. Crystal structures of the kainate receptor GluR5 ligand binding core dimer with novel GluR5-selective antagonists. J Neurosci 2006;26:2852-61.
Plested AJR, Mayer ML. Structure and mechanism of kainate receptor modulation by anions. Neuron 2007;15:539-52.
Structural analysis of NR3 ligand–binding selectivity
The NMDA receptor NR3A subunit is expressed widely in the developing CNS of mammals. Co-assembly of NR3A with NR1 and NR2 modifies NMDA receptor–mediated responses, reducing calcium permeability. In previous work, we characterized the ligand-binding properties of NR3A by using a highly purified, water-soluble NR3A ligand–binding domain. We have now solved high-resolution crystal structures for NR3A complexes with glycine, d-serine, and ACPC and for NR3B complexes with glycine and d-serine. The structures reveal that, despite the substitution by methionine of a large tryptophan residue, which “fills up” the binding pocket in the NR1 subunit and prevents the binding of glutamate, the binding pocket of NR1, NR3A, and NR3B is unusually small compared with other glutamate receptor subtypes that bind to glutamate, indicating that steric occlusion is a common principle for achieving glycine selectivity. In ongoing work, we are investigating a series of mutations based on the crystal structures for NR1 and NR3 subunits to test the hypothesis that subunit-specific differences in ligand affinity result in part from formation of closed-cleft, that is, active conformations
Mayer ML. GRIK4 and the kainate receptor. Am J Psychiatry 2007;164:1148.
Weston MC, Gertler C, Mayer ML, Rosenmund C. Interdomain interactions in AMPA and kainate receptors regulate affinity for glutamate. J Neurosci 2006;26:7650-8.
Weston MC, Schuck P, Ghosal A, Rosenmund C. Conformational restriction blocks glutamate receptor desensitization. Nat Struct Mol Biol 2006;13:1120-7.
Yao Y, Mayer ML. Characterization of a soluble ligand binding domain of the NMDA receptor regulatory subunit NR3A. J Neurosci 2006;26:4559-66.
COLLABORATOR
Phillip 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, Division of Bioengineering and Physical Science, ORS, NIH, Bethesda, MD
For further information, contactmlm@helix.nih.gov.
