Molecular Dynamics Simulations of the Ligand-binding Domain of an N-Methyl-d-aspartate Receptor

Abstract

The mechanism of partial agonism at N-methyl-D-aspartate receptors is an unresolved issue, especially with respect to the role of protein dynamics. We have performed multiple molecular dynamics simulations (7 ؋ 20 ns) to examine the behavior of the ligand-binding core of the NR1 subunit with a series of ligands. Our results show that water plays an important role in stabilizing different conformations of the core and how a closed cleft conformation of the protein might be stabilized in the absence of ligands. In the case of ligand-bound simulations with both full and partial agonists, we observed that ligands within the binding cleft may undergo distinct conformational changes, without grossly influencing the degree of cleft closure within the ligand-binding domain. In agreement with recently published crystallographic data, we also observe similar changes in backbone torsions corresponding to the hinge region between the two lobes for the partial agonist, D-cycloserine. This observation rationalizes the classification of D-cycloserine as a partial agonist and should provide a basis with which to predict partial agonism in this class of receptor by analyzing the behavior of these torsions with other potential ligands. N-Methyl-D-aspartate (NMDA)4 receptors are ligand-gated ion channels that play a major role in learning and memory (1). They have also been implicated in a number of injury or disease states including for example, schizophrenia (2-5). NMDA receptors are a distinct subtype of ionotropic glutamate receptor (6) in that they require both glutamate and glycine for activation and membrane depolarization (7). The receptor is a heterotetrameric cation channel typically comprised of two NR1 subunits that contain the glycine-binding site along with two NR2A-D glutamate-binding sites. The heterogeneity of the complex is further extended if the third subtype (NR3), which also binds glycine, is also taken into account.The architecture of each subunit of NMDA receptors is similar to that of non-NMDA receptors. Each subunit is comprised of an aminoterminal binding domain that shows homology to the LIVBP (leucineisoleucine-valine-binding protein) (8), three transmembrane helices (M1, M2, and M3), a re-entrant P-loop, and a ligand-binding domain that is comprised of two discontinuous polypeptide chains, S1 and S2, that form two distinct lobes or subdomains, D1 and D2 (Fig. 1A). An isolated D1D2 ligand-binding core has been shown to bind ligands with similar affinity as wild-type receptors (9, 10).Recently, the crystal structure of the ligand-binding domain of the NR1 subunit has been solved by x-ray diffraction with both full (9) and partial (10) agonists. The overall fold and structure (Fig. 1B) is very similar to those observed for crystal structures of the ligand-binding domains for the (non-NMDA) GluR2 (11-13), GluR5 (14,15), and GluR6 receptors (Refs. 15 and 16; for recent reviews see Refs. 17-19). The major difference is an insertion of 30 residues into loop 1, which also contains two disulfide bridges. Inter...

Constitutive Activation of the N-Methyl-d-aspartate Receptor via Cleft-spanning Disulfide Bonds

“…One possibility is that stabilization of the closed cleft state, not simply rotation of D2 relative to D1, is the chief determinant of efficacy. This hypothesis finds support in many molecular dynamics studies, which report higher relative domain mobility, decreased hinge stability, and increased agonist flexibility with partial agonists (14,16). Numerous structure/function studies have documented the effects of destabilizing cross-cleft interactions in NR2, GluR2, and GluR6, which include increased deactivation rates as well as lower apparent affinity and efficacy (18 -20).…”

“…Additionally, introduction of a cleft-stabilizing GluR6 D1D2 interaction into GluR2 dramatically slowed deactivation and increased glutamate sensitivity but had no effect on cleft closure, although GluR6 is almost 6°more closed than GluR2 in the full agonist state (18). Molecular dynamics simulations have also observed ligand movement within the NR1 binding pocket without altering the degree of domain closure (16). It has been suggested that both incomplete and unstable cleft closure have similar effects on single channel gating (20), raising the possibility that the degree of cleft closure may simply be a manifestation of closed cleft stability.…”

“…Structural and molecular dynamics data have implicated the conformation of the hinge region, particularly the second interdomain ␤ strand, in sensing agonist efficacy (15,16). Increased interpocket motion has also been associated with NR1 site partial agonism, and both glycine and DCS can reportedly move within the pocket without affecting domain closure (16).…”

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

“…Computer-aided molecular dynamics (MD) simulations that are based on the growing number of LBD structures can be a useful tool to estimate protein fluctuations at physiological temperatures, provided the appropriate caveats are recognized (see section V.A). Such studies have provided hints about permissible intraprotein motions within the LBD during the cleft closure transition as well as interactions at the domain interface (Arinaminpathy et al, 2002;Kaye et al, 2006;Lau and Roux, 2007;Dravid et al, 2010). MD simulations can provide estimates of the stability of the ligand-protein interactions in the binding pocket, adding further detail to our understanding of ligand selectivity and efficacy (Mendieta et al, 2001(Mendieta et al, , 2005Erreger et al, 2005b;Kaye et al, 2006;Pentikä inen et al, 2006;Erreger et al, 2007).…”

“…Such studies have provided hints about permissible intraprotein motions within the LBD during the cleft closure transition as well as interactions at the domain interface (Arinaminpathy et al, 2002;Kaye et al, 2006;Lau and Roux, 2007;Dravid et al, 2010). MD simulations can provide estimates of the stability of the ligand-protein interactions in the binding pocket, adding further detail to our understanding of ligand selectivity and efficacy (Mendieta et al, 2001(Mendieta et al, , 2005Erreger et al, 2005b;Kaye et al, 2006;Pentikä inen et al, 2006;Erreger et al, 2007). Additional experimental techniques that can test predictions obtained from MD simulations or crystal structures include NMR, ultraviolet or infrared spectroscopy, and fluorescence resonance energy transfer Oswald, 2002, 2004;Cheng et al, 2005;Du et al, 2005;Madden et al, 2005;Valentine and Palmer, 2005).…”