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Structural Biology of the Glutamate Receptor
- Mark L. Mayer, PhD, Head, Section on Neurophysiology and Biophysics
- Carla Glasser, PhD, Technical Specialist
- Sagar Chittori, PhD, Visiting Fellow
- Poorva Dharkar, PhD, Visiting Fellow
- Richard Grey, BSc, Postbaccalaureate Fellow
- Austin Zimmet, BSc, Postbaccalaureate Fellow
Ionotropic glutamate receptors (iGluRs) are membrane proteins that act as molecular pores permeable to sodium and calcium ions. iGluRs mediate rapid signal transmission at the majority of excitatory synapses in the mammalian nervous system, converting the pre-synaptic action potential–triggered release of glutamate into a depolarizing post-synaptic potential. The seven iGluR gene families 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, kainate, and NMDA. Given the essential role of iGluRs in normal brain function and development, and mounting evidence that dysfunction of iGluR activity mediates several neurological and psychiatric diseases as well as 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 or by single-molecule cryo-EM provide a framework for designing electro-physiological 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 critical role in brain function.
Key issues in the field include obtaining structures for iGluRs trapped in their three major conformational states, i.e., the resting, activated, and desensitized states, and obtaining insight into the energy landscapes connecting the states. Also of interest are the evolutionary relationships connecting iGluRs in different species and how structurally related chemosensors bind to a wide range of small molecules.
Expression of full-length AMPA and kainate receptor ion channels
Structural studies on CNS membrane proteins are notoriously difficult because commonly used bacterial protein expression systems cannot be employed to express eukaryotic membrane proteins. We succeeded in obtaining highly purified preparations of AMPA receptors and two different kainate receptor subtypes expressed in both insect cells, using baculovirus expression, and in HEK 293 cells, using transient transfection. For both systems, cultures must be grown on the 12-liter and larger scale in order to obtain sufficient protein for biochemical and structural analysis. Because the amounts of protein obtained are still at least an order of magnitude lower than for soluble proteins, it is necessary to use highly efficient methods for establishing conditions in which the proteins remain, for several days following purification, correctly folded and do not aggregate or dissociate into dimers and monomers. To do this, we use tryptophan fluorescence size-exclusion chromatography (FSEC), for which only microgram quantities of purified protein are required for each run of a 300 mM–length analytical gel filtration column. We also investigated receptor stability in different detergent and lipid combinations and in the amphipol A8-35, using FSEC to measure melting curves for protein denaturation as a function of concentration. With this approach and in a collaboration with the Subramaniam lab, we solved 25 Å–resolution structures for GluK2 in resting and desensitized states by cryo-electron tomography.
Structural studies on full-length AMPA and kainate receptors
Our initial studies on GluK2 were limited by poor partitioning of GluK2 into the holes of holey carbon support grids, a common problem that frequently limits structural determination by single-particle cryo-electron microscopy (cryo-EM). This was overcome by depositing, on gold-coated carbon grids, a self-assembled monolayer whose surface properties were controlled by chemical modification. The procedure allowed partitioning of ionotropic glutamate receptors into the holes, thereby enabling higher resolution structural analysis than by using single-particle cryo-EM methods. Using the approach, in collaboration with the Subramaniam lab in NCI, we solved a series of structures for iGluRs trapped in different conformational states, using high-affinity ligands and allosteric modulators to determine how glutamate receptor ion channels accommodate the structural changes necessary for activation and desensitization. For the AMPA receptor GluA2, we solved structures in an antagonist-bound resting state, in a glutamate-bound active state trapped with the allosteric modulator LY451646, and in the desensitized state trapped by the high-affinity full-agonist quisqualic acid. Comparison of the closed- and active-state density maps reveals ligand-binding domain (LBD) “clamshell” closure, as seen for isolated LBD dimers, that produces about 7 Å vertical contraction of the ATD-LBD assembly, measured as a downwards movement at the top of the ATD (amino-terminal domain) tetramer, as well as unanticipated movements in the LBD, in which the dimer pairs rotate about an axis offset from the local axis of two-fold symmetry. This generates a cork screw–like motion, in which the four LBDs expand and likely unwind the iris of the M3 helix bundle closed state.
Analysis of cryo-electron microscopic images for the GluA2–desensitized state revealed evidence of substantial conformational heterogeneity at the ATD layer, precluding determination of a single desensitized-state 3D structure. Three-dimensional classification of the images enabled separation of three dominant classes at nominal resolutions of 21 Å, 23 Å, and 26 Å, with variable degrees of displacement between ATD dimers compared with the closed and active states. In all three classes, the LBD layer separates into four lobes of density, with different degrees of separation between the proximal and distal LBD subunits, strikingly different from the “dimer-of-dimers” structure found in the closed and active states, but reminiscent of the GluK2 structure determined by tomography.
The GluK2–desensitized state was solved to a resolution of 7.6 Å and was adequate to unambiguously show that, in the desensitized state, the ion channel adopts a closed conformation in which the M3 helices form a crossed bundle assembly with the pre-M1 helices wrapped around the outside of the channel, similar to that seen for the antagonist-bound closed state, and revealed for the first time how the LBDs rearrange to permit the ion channel to close even though the individual subunits retain a glutamate-bound closed-cleft active conformation. Desensitization occurs because, in the LBD assembly, the distal subunits swing clockwise by 125° in the horizontal plane, while the proximal subunits rotate by 13°. In the vertical plane, the distal and proximal subunits tilt 11° and 6° away from the global axis of symmetry. As a result of these movements, in the GluK2–desensitized state the LBD layer resembles an inverted pyramid, in which the four subunits are arranged with quasi four-fold symmetry, strikingly different from the two-fold symmetric dimer of dimers assembly in the active state. Overall, the results provide a detailed glimpse into the overall gating cycle of glutamate receptors, an evaluation of the similarities and differences in conformational changes observed in AMPA– and kainate-receptor families and a molecular mechanism for the marked LBD movements that occur during the receptor gating cycle.
Molecular biophysical studies on AMPA–receptor dimer assembly
Sedimentation velocity (SV) analytical ultracentrifugation (AUC) has re-emerged as an important tool for the characterization of biological macromolecules and has been used extensively for analysis of monomer-dimer and dimer-tetramer equilibria for the ATDs and LBDs of iGluRs. Prior reports on AMPA receptor GluA2 dimerization differed in their estimate of the monomer-dimer Kd by a 2,400-fold range, with no consensus as to whether the ATD forms tetramers in solution. In pilot sedimentation velocity (SV) experiments, performed using absorbance and fluorescence detection, we found a narrow range of monomer-dimer Kd values in the low nM range for GluA2, with no detectable formation of tetramers, and no effect of glycosylation or the polypeptide linker connecting the ATDs and LBDs; for GluA3, the monomer-dimer Kd was 5.6 µM, again with no detectable tetramer formation. SV experiments with fluorescence detection for GluA2 labeled with 5,6-carboxyfluorescein (FAM) and fluorescence anisotropy measurements for GluA2 labeled with DyLight405 yielded comparable Kd values; however, the sedimentation coefficients measured by AUC using absorbance differed from those obtained using fluorescence systems (FDS). Given the unique capabilities of the fluorescence detection AUC for future experiments, we conducted an extensive series of experiments to characterize the system, with the goal of identifying procedures that permit accurate Kd measurements at nM protein concentrations. We found by FDS-SV that the FAM label produces a mixed population of receptors with artificially high and low affinity, while DL-488–labeled protein and a GluA2-EGFP fusion protein were monodisperse and had identical Kd values. By performing fluorescence resonance energy transfer (FRET) experiments on GluA2 separately labeled with donor and acceptor dyes, and the kinetics of dimer dissociation measured by the kinetics of quenching following addition of excess unlabeled protein, we obtained independent evidence for perturbation of dimer stability by the FAM label. Thus, in order to take advantage of FDS-SV experiments, control experiments to test for artifacts produced by fluorescent labels are an important consideration. The work is being performed in collaboration with Joy Zhao and Peter Schuck.
Structural studies on glutamate receptors from primitive organisms
In an ongoing series of experiments, a structural and functional analysis is being undertaken in collaboration with the lab of Ela Serpe for glutamate receptor genes in the fruit fly Drosophila, initially on those present at the neuromuscular junction. The results to date reveal a unique ligand-binding profile, the basis of which has been revealed by a crystal structure for one of the ligand-binding domains expressed as a soluble protein in bacteria. Functional analysis reveals strongly voltage-dependent responses, a unique requirement for coexpression of multiple subunits, and permeability to calcium. Related experiments probed glutamate receptors in two ctenophore species, the most primitive animals with a neural net.
- Meyerson JR, Kumar J, Chittori S, Rao P, Pierson J, Bartesaghi A, Mayer ML, Subramaniam S. Structural mechanism of glutamate receptor activation and desensitization. Nature 2014;514:328-334.
- Zhao H, Mayer ML, Schuck P. Analysis of protein interactions with picomolar binding affinity by fluorescence-detected sedimentation velocity. Anal Chem 2014;86:3181-3187.
- Schauder DM, Kuybeda O, Zhang J, Klymko K, Bartesaghi A, Borgnia MJ, Mayer ML, Subramaniam S. Glutamate receptor desensitization is mediated by changes in quaternary structure of the ligand binding domain. Proc Natl Acad Sci USA 2013;110:5921-5926.
- Zhao H, Lomash S, Glasser C, Mayer ML, Schuck P. Analysis of high affinity self-association by fluorescence optical sedimentation velocity analytical ultracentrifugation of labeled proteins: opportunities and limitations. PLoS One 2013;8:e83439.
- Yao Y, Belcher J, Berger AJ, Mayer ML, Lau AY. Conformational analysis of NMDA receptor GluN1, GluN2, and GluN3 ligand-binding domains reveals subtype-specific characteristics. Structure 2013;21:1788-1799.
- Albert Lau, PhD, The Johns Hopkins University School of Medicine, Baltimore, MD
- Peter Schuck, PhD, Laboratory of Cellular Imaging and Macromolecular Biophysics, NIBIB, Bethesda, MD
- Mihaela Serpe, PhD, Program in Cellular Regulation and Metabolism, NICHD, Bethesda, MD
- Sriram Subramaniam, PhD, Laboratory of Cell Biology, Center for Cancer Research, NCI, Bethesda, MD
- Joy Zhao, PhD, Laboratory of Cellular Imaging and Macromolecular Biophysics, NIBIB, Bethesda, MD
For more information, email email@example.com or visit snb.nichd.nih.gov or neuroscience.nih.gov/Faculty/Profile/mark-mayer.aspx.