Structural Mechanism of N-Methyl-D-Aspartate Receptor Type 1 Partial Agonism

Citation: Ylilauri M, Pentikainen OT ( Structural Mechanism of N -Methyl-D-Aspartate Receptor Type 1 Partial Agonism Mikko Ylilauri 0 Olli T. Pentika inen 0 Andrew Jenkins, Emory University, United States of America 0 Computational Bioscience Laboratory, Department of Biological and Environmental Science & Nanoscience Center, University of Jyva skyla , Jyva skyla , Finland N-methyl-D-aspartate (NMDA) receptors belong to a family of ionotropic glutamate receptors that contribute to the signal transmission in the central nervous system. NMDA receptors are heterotetramers that usually consist of two GluN1 and GluN2 monomers. The extracellular ligand-binding domain (LBD) of a monomer is comprised of discontinuous segments that form the functional domains D1 and D2. While the binding of a full agonist glycine to LBD of GluN1 is linked to cleft closure and subsequent ion-channel opening, partial agonists are known to activate the receptor only sub-maximally. Although the crystal structures of the LBD of related GluA2 receptor explain the mechanism for the partial agonism, structures of GluN1-LBD cannot distinguish the difference between full and partial agonists. It is, however, probable that the partial agonists of GluN1 alter the structure of the LBD in order to result in a different pharmacological response than seen with full agonists. In this study, we used molecular dynamics simulations to reveal an intermediate closure-stage for GluN1, which is unseen in crystal structures. According to our calculations, this intermediate closure is not a transient stage but an energetically stable conformation. Our results demonstrate that the partial agonist cannot exert firm GluN1-LBD closure, especially if there is even a small force that disrupts the LBD closure. Accordingly, this result suggests the importance of forces from the ion channel for the relationship between pharmacological response and the structure of the LBD of members of this receptor family. - Funding: This study was funded by the National Doctoral Programme in Informational and Structural Biology (M.Y.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. N-methyl-D-aspartate receptors (NMDARs) belong to a family of ionotropic glutamate receptors (iGluRs) that contribute to signal transmission in the central nervous system [1]. NMDARs play crucial roles in learning and synaptic plasticity, for example [2], [3], [4]. All the iGluRs have been implicated in various diseases, especially neurological disorders. Disease states linked to NMDARs include Parkinsons disease, schizophrenia and stroke, among others [5], [6]. Similar to GluA2 (Fig. 1A), NMDAR probably is a heterotetramer that usually consists of two GluN1 (NMDA-R1) and GluN2 (NMDA-R2) monomers [7]. The functional heterogeneity of NMDARs arises from a wide variety of GluN2 subunits (for a recent review, see [8]). The ligandbinding domain (LBD) of iGluRs is comprised of discontinuous segments that form the functional domains 1 and 2 (D1 and D2) [9]. Although the recombinant LBD forms only part of the iGluR monomer, it shows a similar ligand-binding affinity to that of wildtype receptors [10], [11], [12]. Thus, this domain has been widely applied in crystallography, for example [11], [12], [13], [14], [15], [16] (Fig. 1BC). Full agonists provoke full LBD closure, leading to opening of the ion channel [13]. In contrast to the AMPA-selective glutamate receptor 2 (GluA2; GluR2) where partial agonists wedge the LBD into a moderately closed state [13], [17] (Fig. 1B), the crystal structures of GluN1 imply that the partial agonists induce full receptor closure [11] (Fig. 1C), pointing to a different mechanism. This view was supported by a recent study [18] that used luminescence resonance energy transfer (LRET) to measure the extent of cleft closure in GluN1. No difference was found between the closure stages of full or partial agonist bound GluN1LBD. Interestingly, however, in the same study, GluN2-LBD exhibited an intermediate cleft closure when bound to a partial agonist. In addition to many crystallization studies, the ligand binding and closure of the iGluR-LBD have been explored using various experimental methods, including electrophysiology [12], [19], [20], fluorescence resonance energy transfer [21], and radioligand binding [16]. In addition to these experimental approaches, several recent studies have also exploited sophisticated computational methods to examine the structure and function of iGluRs. In particular, molecular dynamics (MD) simulations have been utilized to study the motion of receptor and ligand-receptor interactions occurring in solvent [22], [23]. For example, the role of water molecules inside the ligand-binding cleft [24], the pharmacology of novel ligands [25], and the subtype selectivity of antagonist ligands [26] have been studied with the help of this in silico method. However, closing an open-cleft receptor with a bound ligand has been reached computationally thus far only when exploited with biased MD simulations, for example the umbrella sampling method [27]. The antagonism of NMDA receptors has been widely studied for possible treatment of many neurological disorders [5], [28]. However, it has been proposed that partial agonists could be more advantageous as therapeutics because of their capability to permit some level of normal synaptic transmission while simultaneously suppressing excessive activation [29], [30], [31]. In fact, it has recently become evident that GluN1-specific partial agonists could be used to treat autism, for example (see [32] for review). However, although a growing number of studies concerning partial agonism of NMDA receptors have been published (see for example [12], [20], [33], [34]), only a few have examined the structure and motion of the LBD and its interactions with the ligand at the atomic level [22], [35], [36]. We have previously shown in MD simulations that the GluN1LBD is able to adjust to more open conformations than crystallization studies have shown [36]. In addition, we have suggested that the stability of the cleft closure is associated with partial agonism. Incomplete closure of the GluN1-LBD with a bound partial agonist is not only interesting but also highly important pharmacologically. Indeed, it has been shown that the intrasubunit movements at linkers between LBD and transmembrane (TM) region are tightly coupled across the four subunits of NMDAR [37]. Thus, the binding of partial agonist molecules to two GluN1 subunits of the tetrameric receptor, which leads to incomplete closure of the LBD, would prevent full ion channel opening despite simultaneous full agonist binding to two GluN2 subunits. In the present study, various computational methods were utilized in order to obtain a detailed view of the interactions tak (...truncated)