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  • br NMDA receptor LBDs The study of GluA was

    2022-07-04


    NMDA receptor LBDs The study of GluA2 was later expanded to include NMDA receptors [22]. NMDA receptors are obligate heterotetramers that are composed of two glutamate-binding subunits and two glycine-, or d-serine-, binding subunits. In this study, crystal structures of the NMDA receptor glycine-binding domains GluN1 and GluN3A in the apo state were reported, where GluN1 adopts an open conformation while GluN3A adopts a closed one. Umbrella sampling free LY364947 computations involving the LBDs of GluN1, GluN2A, and GluN3A show these domains can easily access both open and closed conformations in the apo state. Based on the PMFs, the GluN LBDs, especially the glycine-binding subunits, seem to bind agonist via a conformational selection mechanism [23,24]. The GluA2 LBD, on the other hand, seems to bind agonist via an induced fit mechanism [25]. It is noted, however, that these mechanisms are not mutually exclusive [26], and perhaps models that define mechanisms of binding and conformational change in less rigid terms should be considered [27]. As with the GluA2 LBD, the computed PMF for GluN1 was found to be consistent with smFRET measurements [28]. An analysis of conformational dynamics indicates that the NMDA and AMPA receptor LBDs have non-uniform modes of large-scale motion. This spectrum of conformational dynamics may have functional consequences within an intact receptor. The nature of partial agonism in NMDA receptors has been investigated using PMFs computed in similar fashion as above [29]. PMFs computed for the GluN1 LBD bound to a variety of ligands suggest curvature of a free energy surface may be an indicator of whether the bound ligand is a partial agonist or a full agonist, with partial agonists exhibiting reduced curvature compared with full agonists. Stationary gating in NMDA receptors has also been studied using computed PMFs [30]. Stationary gating refers to the phenomenon of stochastic opening and closing of the ion channel when agonist is present at saturating concentrations. Experimentally this can be reproduced by disulfide crosslinking the two lobes of the LBD, closely mimicking bound agonist [31]. PMFs of agonist-bound GluN1 and GluN2A, and their crosslinked forms, suggest both agonist-bound and crosslinked LBDs sample semi-closed conformations. It is proposed that during stationary gating, the channel closes when the LBDs occupy semi-closed conformations. Evolutionary precursors to NMDA receptors include glycine-activated receptors found in ctenophore genomes [[32], [33], [34]]. The ctenophore glycine receptors feature a distinct salt bridge within the LBD that bridges the two lobes, a “molecular lock”, that has not been found in iGluRs of other organisms, including mammals. A study combining crystallographic, biochemical, electrophysiological, and computational methods to elucidate the role of this molecular lock in a ctenophore iGluR was carried out [35]. Perturbations to the lock were demonstrated to tune receptor kinetics and thermodynamics over very broad ranges. This is contrary to previous studies of perturbations in interdomain contacts in vertebrate iGluRs, which revealed only modest tuning of ligand affinity. Free energy calculations show that the lock is strategically positioned to maximally impede the egress of agonist molecules from the binding pocket, which may be the basis for the LBD's extraordinarily high affinity for glycine.
    Optogenetics Photoswitched tethered ligands (PTLs) can be covalently attached to iGluR LBDs to control receptor function remotely with light [[36], [37], [38]]. One such PTL belonging to the photoisomerizable maleimide-azobenzene-glutamate (MAG) family was found to activate GluK2 (iGluR6) when the ligand's linker is in the cis state at some attachment sites, but in trans at others [39]. Conformational free energy simulations that took into account both ligand docking and LBD closure explained the experimental observation. Receptor activation via photoswitching of MAG was found to depend on two factors: (i) the population of MAG conformers that enable the glutamate moiety of the ligand to insert into the binding site of the open LBD and (ii) the extent of LBD closure that is allowed given the linker's conformation and attachment site. An umbrella sampling study of the GluK2 LBD bound to gluazo, a photo switchable ligand, has also been carried out [40].