Abstract
The prevailing structural model for ligand activation of ionotropic glutamate receptors posits that agonist efficacy arises from the stability and magnitude of induced domain closure in the ligand-binding core structure. Here we describe an exception to the correlation between ligand efficacy and domain closure. A weakly efficacious partial agonist of very low potency for homomeric iGluR5 kainate receptors, 8,9-dideoxyneodysiherbaine (MSVIII-19), induced a fully closed iGluR5 ligand-binding core. The degree of relative domain closure, approximately 30 degrees , was similar to that we resolved with the structurally related high affinity agonist dysiherbaine and to that of l-glutamate. The pharmacological activity of MSVIII-19 was confirmed in patch clamp recordings from transfected HEK293 cells, where MSVIII-19 predominantly inhibits iGluR5-2a, with little activation apparent at a high concentration (1 mm) of MSVIII-19 (<1% of mean glutamate-evoked currents). To determine the efficacy of the ligand quantitatively, we constructed concentration-response relationships for MSVIII-19 following potentiation of steady-state currents with concanavalin A (EC(50) = 3.6 microm) and on the nondesensitizing receptor mutant iGluR5-2b(Y506C/L768C) (EC(50) = 8.1 microm). MSVIII-19 exhibited a maximum of 16% of full agonist efficacy, as measured in parallel recordings with glutamate. Molecular dynamics simulations and electrophysiological recordings confirm that the specificity of MSVIII-19 for iGluR5 is partly attributable to interdomain hydrogen bond residues Glu(441) and Ser(721) in the iGluR5-S1S2 structure. The weaker interactions of MSVIII-19 with iGluR5 compared with dysiherbaine, together with altered stability of the interdomain interaction, may be responsible for the apparent uncoupling of domain closure and channel opening in this kainate receptor subunit.
Highlights
The prevailing structural model for ligand activation of ionotropic glutamate receptors posits that agonist efficacy arises from the stability and magnitude of induced domain closure in the ligand-binding core structure
The Ionotropic glutamate receptors (iGluRs) consist of three different classes of receptors, N-methyl-D-aspartic acid (NMDA), ␣-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors [1], which are assembled as tetramers in a dimer of dimers configuration [2, 3]
The binding affinities of DH and MSVIII-19 were determined for the iGluR5-S1S2 construct by competition studies with 3H-labeled (2S,4R)-4-methyl-glutamic acid to ensure that the compounds bind with comparable affinities to the construct and the full-length iGluR5 receptor
Summary
X-ray Structure Determination—The iGluR5-S1S2 construct developed by Naur et al was used, and the protein was expressed and purified essentially as reported [17]. The Kd value for iGluR52a(S721N) was determined in saturation isotherm experiments with varying concentrations of [3H]kainate under the same conditions as those in the displacement assays These data were plotted and fit with a one-site binding curve (hyperbola) using Prism 4. Tleap in Antechamber-1.27 [33] was used to (a) set the force field parameters for the protein (parm99) and ligands (gaff), (b) add hydrogen atoms, (c) neutralize the system with chloride ions for iGluR5 and sodium ions for iGluR6, and (d) solvate the system with a rectangular box (13 Å in every dimension) of transferable intermolecular potential three-point water molecules (TIP3P). The water molecules, counterions, and amino acid side chains were minimized with the conjugate gradient algorithm (3000 steps) as the rest of the complex was constrained by restraining C␣ atoms into their initial positions with harmonic force of 5 kcal molϪ1 ÅϪ2.
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