Structural Determinants of Agonist Efficacy at the Glutamate Binding Site ofN-Methyl-d-Aspartate Receptors

Abstract

N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels assembled from GluN1 and GluN2 subunits. We used a series of N-hydroxypyrazole-5-glycine (NHP5G) partial agonists at the GluN2 glutamate binding site as tools to study activation of GluN1/GluN2A and GluN1/GluN2D NMDA receptor subtypes. Using two-electrode voltage-clamp electrophysiology, fast-application patch-clamp, and single-channel recordings, we show that propyl-and ethyl-substituted NHP5G agonists have a broad range of agonist efficacies relative to the full agonist glutamate (,1-72%). Crystal structures of the agonist binding domains (ABDs) of GluN2A and GluN2D do not reveal any differences in the overall domain conformation induced by binding of the full agonist glutamate or the partial agonist propyl-NHP5G, which is strikingly different from ABD structures of 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propanoate (AMPA) and kainate receptors bound to full and partial agonists. Subsequent evaluation of relative NHP5G agonist efficacy at GluN2A-GluN2D chimeric subunits implicates the amino-terminal domain (ATD) as a strong determinant of agonist efficacy, suggesting that interdomain interactions between the ABD and the ATD may be a central element in controlling the manner by which agonist binding leads to channel opening. We propose that variation in the overall receptor conformation, which is strongly influenced by the nature of interdomain interactions in resting and active states, mediates differences in agonist efficacy and partial agonism at the GluN2 subunits.

Semiclosed Conformations of the Ligand-Binding Domains of NMDA Receptors during Stationary Gating

“…For AMPA receptor LBDs, the degree of agonist-induced cleft closure is correlated with agonist efficacy (14,15,24). On the other hand, for NMDA receptor LBDs, both full agonists and a number of partial agonists induce similar degrees of cleft closure, whereas antagonists keep the cleft open (16,(18)(19)(20)22). These results indicate that the two subtypes of FIGURE 1 Modular architecture of an NMDA receptor.…”

“…The structures of isolated LBDs from various iGluR subunits have been determined by x-ray crystallography in an assortment of ligand-bound forms (2,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). For AMPA receptor LBDs, the degree of agonist-induced cleft closure is correlated with agonist efficacy (14,15,24).…”

Structural Determinants and Mechanism of Action of a GluN2C-selective NMDA Receptor Positive Allosteric Modulator

“…The relatively fast time course for the onset and recovery from positive allosteric modulation suggests that the extracellular binding site for PYD is readily accessible. Moreover, the region between the ATD and LBD is well positioned to influence a wide range of channel properties including agonist EC 50 , deactivation time course and open probability (Gielen et al, 2009;Yuan et al, 2009;Hansen et al, 2013).…”

“…Xenopus laevis oocytes were obtained from EcoCyte (Austin, TX) injected with cRNA created by in vitro transcription using the mMessage mMachine kits according to the manufacturer's instructions (Life Technologies/Ambion, Grand Island, NY) as previously described (Hansen et al, 2013). Plasmids containing the genes for the GABA A (a1b2g2s), GABA C (r1), glycine (a1), serotonin (5-HT 3A ), nicotinic acetylcholine receptor (a1b1dg, a2b4, a4b3, a9a10), and purinergic (P2X 2 rat, P2X 2 human) receptors were provided by Drs.…”

Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors

“…The small volume may pose an important size constraint for future design efforts of new GluD2 agonists. Thus, creation of agonists with improved affinity by addition Ligands Modulating Activity of the GluD2 Lurcher Mutant of larger functional groups to the scaffold of the endogenous agonist, as has been found possible for analogs of L-glutamate at AMPA (Vogensen et al, 2011;Juknaite et al, 2012), NMDA (Erreger et al, 2005;Clausen et al, 2008;Hansen et al, 2013), and kainate receptors (Zhou et al, 1997;Juknaite et al, 2012), may prove difficult for GluD2. This is reflected in the present ligand screen where the only D-Ser analogs that preserve activity are those in which the core functional groups are replaced with functionalities of similar size ( Fig.…”