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

Abstract

NMDA receptors are tetrameric ligand-gated ion channels. In the continuous presence of saturating agonists, NMDA receptors undergo stationary gating, in which the channel stochastically switches between an open state that permits ion conductance and a closed state that prevents permeation. The ligand-binding domains (LBDs) of the four subunits are expected to have closed clefts in the channel-open state. On the other hand, there is little knowledge about the conformational status of the LBDs in the channel-closed state during stationary gating. To probe the latter conformational status, Kussius and Popescu engineered interlobe disulfide cross-links in NMDA receptors and found that the cross-linking produced stationary gating kinetics that differed only subtly from that produced by agonist binding. These authors assumed that the cross-linking immobilized the LBDs in cleft-closed conformations, and consequently concluded that throughout stationary gating, agonistbound LBDs also stayed predominantly in cleft-closed conformations and made only infrequent excursions to cleft-open conformations. Here, by calculating the conformational free energies of cross-linked and agonist-bound LBDs, we assess whether cross-linking actually traps the LBDs in cleft-closed conformations and delineate semiclosed conformations of agonist-bound LBDs that may potentially be thermodynamically and kinetically important during stationary gating. Our free-energy results show that the cross-linked LBDs are not locked in the fully closed form; rather, they sample semiclosed conformations almost as readily as the agonist-bound LBDs. Several lines of reasoning suggest that LBDs are semiclosed in the channel-closed state during stationary gating. Our free-energy simulations suggest possible structural details of such semiclosed LBD conformations, including intra-and intermolecular interactions that serve as alternatives to those in the cleft-closed conformations.

Allosteric Interactions between NMDA Receptor Subunits Shape the Developmental Shift in Channel Properties

“…Since NMDAR activation requires agonist binding to all four subunits (Benveniste and Mayer, 1991), the receptor deactivation rate in the continuous presence of glycine may be defined largely by the GluN2 subunit with fastest glutamate unbinding. To address this assumption and to conclusively test whether interactions occur between GluN2A and GluN2B subunits, we introduced two cysteine mutations into GluN2, which spontaneously form a disulfide bond that locks the ligand binding domain (LBD) in a conformation similar to the glutamate-bound state (GluN2A: K487C + N687C; GluN2B: K488C + N688C; hereafter Acc and Bcc, respectively; Blanke and VanDongen, 2008; Dai and Zhou, 2016; Kussius and Popescu, 2010). Thus, this disulfide bond will generate “constitutively liganded” GluN2 subunits with high efficiency (Fig.…”

Kinetic models for activation and modulation of NMDA receptor subtypes

“…Early evidence from measurements of intramolecular motions in functional receptors subject to allosteric inhibition [44,45] and from molecular dynamics simulations ([46] and see below) largely support this hypothesis. However, the information necessary to begin assigning even rough correspondence between kinetic states and families of structural conformations is currently insufficient.…”

NMDA receptors: linking physiological output to biophysical operation

“…Furthermore, computational dynamics modelling based on the available NMDA receptor structures will provide more detailed structural models of activation 128,134 . As longer time scales become accessible this approach promises to bridge the present gap between structural and kinetic models of gating.…”