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• W2806876216 abstract "•Glutamate binds to NMDA receptors via a guided-diffusion mechanism•Glycine binds to NMDA receptors via an unguided-diffusion mechanism•All-atom simulations locate metastable sites that assist glutamate binding•Binding of glutamate can occur in two orientations At central nervous system synapses, agonist binding to postsynaptic ionotropic glutamate receptors (iGluRs) results in signaling between neurons. N-Methyl-D-aspartic acid (NMDA) receptors are a unique family of iGluRs that activate in response to the concurrent binding of glutamate and glycine. Here, we investigate the process of agonist binding to the GluN2A (glutamate binding) and GluN1 (glycine binding) NMDA receptor subtypes using long-timescale unbiased molecular dynamics simulations. We find that positively charged residues on the surface of the GluN2A ligand-binding domain (LBD) assist glutamate binding via a “guided-diffusion” mechanism, similar in fashion to glutamate binding to the GluA2 LBD of AMPA receptors. Glutamate can also bind in an inverted orientation. Glycine, on the other hand, binds to the GluN1 LBD via an “unguided-diffusion” mechanism, whereby glycine finds its binding site primarily by random thermal fluctuations. Free energy calculations quantify the glutamate- and glycine-binding processes. At central nervous system synapses, agonist binding to postsynaptic ionotropic glutamate receptors (iGluRs) results in signaling between neurons. N-Methyl-D-aspartic acid (NMDA) receptors are a unique family of iGluRs that activate in response to the concurrent binding of glutamate and glycine. Here, we investigate the process of agonist binding to the GluN2A (glutamate binding) and GluN1 (glycine binding) NMDA receptor subtypes using long-timescale unbiased molecular dynamics simulations. We find that positively charged residues on the surface of the GluN2A ligand-binding domain (LBD) assist glutamate binding via a “guided-diffusion” mechanism, similar in fashion to glutamate binding to the GluA2 LBD of AMPA receptors. Glutamate can also bind in an inverted orientation. Glycine, on the other hand, binds to the GluN1 LBD via an “unguided-diffusion” mechanism, whereby glycine finds its binding site primarily by random thermal fluctuations. Free energy calculations quantify the glutamate- and glycine-binding processes. N-Methyl-D-aspartic acid (NMDA) receptors are ligand-gated ion channels important for fast excitatory synaptic transmission, distributed throughout the central nervous system (Moriyoshi et al., 1991Moriyoshi K. Masu M. Ishii T. Shigemoto R. Mizuno N. Nakanishi S. Molecular cloning and characterization of the rat NMDA receptor.Nature. 1991; 354: 31-37Crossref PubMed Scopus (1624) Google Scholar, Monyer et al., 1992Monyer H. Sprengel R. Schoepfer R. Herb A. Higuchi M. Lomeli H. Burnashev N. Sakmann B. Seeburg P.H. Heteromeric NMDA receptors: molecular and functional distinction of subtypes.Science. 1992; 256: 1217-1221Crossref PubMed Scopus (2256) Google Scholar, Paoletti et al., 2013Paoletti P. Bellone C. Zhou Q. NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease.Nat. Rev. Neurosci. 2013; 14: 383-400Crossref PubMed Scopus (1506) Google Scholar). They are necessary for learning and memory and are drug targets for treating Alzheimer's disease (McKeage, 2009McKeage K. Memantine: a review of its use in moderate to severe Alzheimer’s disease.CNS Drugs. 2009; 23: 881-897Crossref PubMed Scopus (70) Google Scholar), depression (Moskal et al., 2014Moskal J.R. Burch R. Burgdorf J.S. Kroes R.A. Stanton P.K. Disterhoft J.F. Leander J.D. GLYX-13, an NMDA receptor glycine site functional partial agonist enhances cognition and produces antidepressant effects without the psychotomimetic side effects of NMDA receptor antagonists.Expert Opin. Investig. Drugs. 2014; 23: 243-254Crossref PubMed Scopus (102) Google Scholar), epilepsy (Hu et al., 2016Hu C. Chen W. Myers S.J. Yuan H. Traynelis S.F. Human GRIN2B variants in neurodevelopmental disorders.J. Pharmacol. Sci. 2016; 132: 115-121Crossref PubMed Scopus (133) Google Scholar), and schizophrenia (Balu, 2016Balu D.T. The NMDA receptor and schizophrenia.Adv. Pharmacol. 2016; 76: 351-382Crossref PubMed Scopus (152) Google Scholar). These receptors are obligate heterotetramers, typically comprising two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits (Mayer et al., 1984Mayer M.L. Westbrook G.L. Guthrie P.B. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones.Nature. 1984; 309: 261-263Crossref PubMed Scopus (2216) Google Scholar). Each subunit contains an extracellular amino-terminal domain (ATD) and ligand-binding domain (LBD) in addition to a transmembrane domain (TMD) and an intracellular C-terminal domain (CTD) (Mayer, 2017Mayer M.L. The challenge of interpreting glutamate-receptor ion-channel structures.Biophys. J. 2017; 113: 2143-2151Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The LBDs possess an overall clamshell-like conformation in which a hinge separates two lobes of the LBD (lobes 1 and 2) (Figure 1). The TMDs of the four subunits surround a central pore to form the core of the ion channel, and in each subunit, the TMD is connected to lobe 2 of the LBD by three short linkers. Binding of two distinct agonists, glutamate and glycine (or D-serine), to the individual glutamate- and glycine-binding LBDs activates the channel. Agonist binding triggers conformational change in the LBD, causing the two lobes to close around the ligand and is thought to provide the useful work to open the channel pore, which, together with a voltage-dependent unblock of magnesium, allows the entry of calcium into the postsynaptic cell and depolarization of the membrane potential (Mayer et al., 1984Mayer M.L. Westbrook G.L. Guthrie P.B. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones.Nature. 1984; 309: 261-263Crossref PubMed Scopus (2216) Google Scholar, Nowak et al., 1984Nowak L. Bregestovski P. Ascher P. Herbet A. Prochiantz A. Magnesium gates glutamate-activated channels in mouse central neurones.Nature. 1984; 307: 462-465Crossref PubMed Scopus (3093) Google Scholar, MacDermott et al., 1986MacDermott A.B. Mayer M.L. Westbrook G.L. Smith S.J. Barker J.L. NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones.Nature. 1986; 321: 519-522Crossref PubMed Scopus (1375) Google Scholar). Crystallographic and cryo-electron microscopic studies have captured NMDA receptor LBDs bound to glutamate and glycine, as well as various other agonists and antagonists, shedding light on the molecular interactions involved in stabilizing the ligands and conformational changes accompanying ligand binding (Inanobe et al., 2005Inanobe A. Furukawa H. Gouaux E. Mechanism of partial agonist action at the NR1 subunit of NMDA receptors.Neuron. 2005; 47: 71-84Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, Furukawa et al., 2005Furukawa H. Singh S.K. Mancusso R. Gouaux E. Subunit arrangement and function in NMDA receptors.Nature. 2005; 438: 185-192Crossref PubMed Scopus (603) Google Scholar, Yao et al., 2013Yao Y. Belcher J. Berger A.J. Mayer M.L. Lau A.Y. Conformational analysis of NMDA receptor GluN1, GluN2, and GluN3 ligand-binding domains reveals subtype-specific characteristics.Structure. 2013; 21: 1788-1799Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, Jespersen et al., 2014Jespersen A. Tajima N. Fernandez-Cuervo G. Garnier-Amblard E.C. Furukawa H. Structural insights into competitive antagonism in NMDA receptors.Neuron. 2014; 81: 366-378Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, Hackos et al., 2016Hackos D.H. Lupardus P.J. Grand T. Chen Y. Wang T.-M. Reynen P. Gustafson A. Wallweber H.J.A. Volgraf M. Sellers B.D. et al.Positive allosteric modulators of GluN2A-containing NMDARs with distinct modes of action and impacts on circuit function.Neuron. 2016; 89: 983-999Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, Volgraf et al., 2016Volgraf M. Sellers B.D. Jiang Y. Wu G. Ly C.Q. Villemure E. Pastor R.M. Yuen P. Lu A. Luo X. et al.Discovery of GluN2A-selective NMDA receptor positive allosteric modulators (PAMs): tuning deactivation kinetics via structure-based design.J. Med. Chem. 2016; 59: 2760-2779Crossref PubMed Scopus (68) Google Scholar, Zhu et al., 2016Zhu S. Stein R.A. Yoshioka C. Lee C.-H. Goehring A. Mchaourab H.S. Gouaux E. Mechanism of NMDA receptor inhibition and activation.Cell. 2016; 165: 704-714Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, Tajima et al., 2016Tajima N. Karakas E. Grant T. Simorowski N. Diaz-Avalos R. Grigorieff N. Furukawa H. Activation of NMDA receptors and the mechanism of inhibition by ifenprodil.Nature. 2016; 534: 63-68Crossref PubMed Scopus (137) Google Scholar, Yi et al., 2016Yi F. Mou T.-C. Dorsett K.N. Volkmann R.A. Menniti F.S. Sprang S.R. Hansen K.B. Structural basis for negative allosteric modulation of GluN2A-containing NMDA receptors.Neuron. 2016; 91: 1316-1329Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, Villemure et al., 2017Villemure E. Volgraf M. Jiang Y. Wu G. Ly C.Q. Yuen P. Lu A. Luo X. Liu M. Zhang S. et al.GluN2A-selective pyridopyrimidinone series of NMDAR positive allosteric modulators with an improved in vivo profile.ACS Med. Chem. Lett. 2017; 8: 84-89Crossref PubMed Scopus (23) Google Scholar, Lü et al., 2017Lü W. Du J. Goehring A. Gouaux E. Cryo-EM structures of the triheteromeric NMDA receptor and its allosteric modulation.Science. 2017; 355 (Published online February 23, 2017)https://doi.org/10.1126/science.aal3729Crossref Scopus (101) Google Scholar, Romero-Hernandez and Furukawa, 2017Romero-Hernandez A. Furukawa H. Novel mode of antagonist binding in NMDA receptors revealed by the crystal structure of the GluN1-GluN2A ligand-binding domain complexed to NVP-AAM077.Mol. Pharmacol. 2017; 92: 22-29Crossref PubMed Scopus (20) Google Scholar, Lind et al., 2017Lind G.E. Mou T.-C. Tamborini L. Pomper M.G. Micheli C.D. Conti P. Pinto A. Hansen K.B. Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits.Proc. Natl. Acad. Sci. USA. 2017; 114: E6942-E6951Crossref PubMed Scopus (27) Google Scholar). The precise molecular details of the dynamical processes associated with ligand binding, however, are unknown. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are related ionotropic glutamate receptors (iGluRs) that mediate fast excitatory neurotransmission, and are also typically heteromeric ion channels, composed of GluA2 subunits in conjugation with GluA1, GluA3, or GluA4 subunits (Isaac et al., 2007Isaac J.T.R. Ashby M.C. McBain C.J. The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity.Neuron. 2007; 54: 859-871Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, Herguedas et al., 2016Herguedas B. García-Nafría J. Cais O. Fernández-Leiro R. Krieger J. Ho H. Greger I.H. Structure and organization of heteromeric AMPA-type glutamate receptors.Science. 2016; 352: aad3873Crossref PubMed Scopus (80) Google Scholar). Like NMDA receptors, they have functionally modular domains separated into the ATDs, LBDs, TMD, and CTDs, but differ in that activation of the channel requires binding of only a single agonist, glutamate, to the separate LBDs. Recent computational and electrophysiology studies have elucidated the binding mechanisms of glutamate to the AMPA receptor and found binding intermediates that assist the process of ligand binding (Yu and Lau, 2017Yu A. Lau A.Y. Energetics of glutamate binding to an ionotropic glutamate receptor.J. Phys. Chem. B. 2017; 121: 10436-10442Crossref PubMed Scopus (10) Google Scholar, Yu et al., 2018Yu A. Salazar H. Plested A.J.R. Lau A.Y. Neurotransmitter funneling optimizes glutamate receptor kinetics.Neuron. 2018; 97: 139-149.e4Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Molecular simulation has proven useful in describing the dynamics and energetics of NMDA receptor LBD cleft closure and the potentiating role of glycans (Yao et al., 2013Yao Y. Belcher J. Berger A.J. Mayer M.L. Lau A.Y. Conformational analysis of NMDA receptor GluN1, GluN2, and GluN3 ligand-binding domains reveals subtype-specific characteristics.Structure. 2013; 21: 1788-1799Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, Dai and Zhou, 2013Dai J. Zhou H.-X. An NMDA receptor gating mechanism developed from MD simulations reveals molecular details underlying subunit-specific contributions.Biophys. J. 2013; 104: 2170-2181Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, Dai and Zhou, 2015Dai J. Zhou H.-X. Reduced curvature of ligand-binding domain free-energy surface underlies partial agonism at NMDA receptors.Structure. 2015; 23: 228-236Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, Dai and Zhou, 2016Dai J. Zhou H.-X. Semiclosed conformations of the ligand-binding domains of NMDA receptors during stationary gating.Biophys. J. 2016; 111: 1418-1428Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, Dutta et al., 2015Dutta A. Krieger J. Lee J.Y. Garcia-Nafria J. Greger I.H. Bahar I. Cooperative dynamics of intact AMPA and NMDA glutamate receptors: similarities and subfamily-specific differences.Structure. 2015; 23: 1692-1704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, Omotuyi and Ueda, 2015Omotuyi O.I. Ueda H. Molecular dynamics study-based mechanism of nefiracetam-induced NMDA receptor potentiation.Comput. Biol. Chem. 2015; 55: 14-22Crossref PubMed Scopus (14) Google Scholar, Sinitskiy and Pande, 2017Sinitskiy A.V. Pande V.S. Simulated dynamics of glycans on ligand-binding domain of NMDA receptors reveals strong dynamic coupling between glycans and protein core.J. Chem. Theory Comput. 2017; 13: 5496-5505Crossref PubMed Scopus (14) Google Scholar, Sinitskiy et al., 2017Sinitskiy A.V. Stanley N.H. Hackos D.H. Hanson J.E. Sellers B.D. Pande V.S. Computationally discovered potentiating role of glycans on NMDA receptors.Sci. Rep. 2017; 7: 44578Crossref PubMed Scopus (19) Google Scholar). Here, using an aggregate of ∼75 μs of unbiased all-atom molecular dynamics (MD) simulations, we investigate the molecular mechanisms of binding for both glutamate and glycine to the LBDs of the NMDA receptor subunits, GluN2A and GluN1. We directly simulate glutamate binding to the GluN2A LBD, as well as glycine binding to the GluN1 LBD, and subsequent cleft closure. Analysis of the cross-lobe interactions formed during cleft closure suggests these interactions may contribute to previously observed dynamical motions (Yao et al., 2013Yao Y. Belcher J. Berger A.J. Mayer M.L. Lau A.Y. Conformational analysis of NMDA receptor GluN1, GluN2, and GluN3 ligand-binding domains reveals subtype-specific characteristics.Structure. 2013; 21: 1788-1799Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Free energy landscapes indicate that the dynamic mechanisms responsible for glutamate binding are distinct from that of glycine binding in NMDA receptors. Previously, glutamate was found to bind to the GluA2 LBD via a series of metastable interactions in which positively charged sidechains help guide the ligand into its binding pocket (Yu and Lau, 2017Yu A. Lau A.Y. Energetics of glutamate binding to an ionotropic glutamate receptor.J. Phys. Chem. B. 2017; 121: 10436-10442Crossref PubMed Scopus (10) Google Scholar, Yu et al., 2018Yu A. Salazar H. Plested A.J.R. Lau A.Y. Neurotransmitter funneling optimizes glutamate receptor kinetics.Neuron. 2018; 97: 139-149.e4Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Glutamate binding to the GluN2A LBD proceeds in a similar fashion (Figures 1 and 2). We simulated five trajectories involving GluN2A and 40 mM glutamate (23.5 μs), five trajectories involving GluN2A and 4 mM glutamate (11.5 μs), and three trajectories involving a GluN2A/GluN1 heterodimer with the following ligand concentrations: 20 mM glutamate and 20 mM glycine (5.2 μs), 20 mM glutamate and 18 mM glycine (5.3 μs), and 2 mM glutamate and 2 mM glycine (4.2 μs). In the heterodimer simulations, glutamate bound seven times to the GluN2A LBD, but glycine binding to the GluN1 LBD was not observed. In trajectory T1 (Video S1), the glutamate ligand first contacts the LBD on the ξ1 side of the binding cleft (Figure 2B). The α-carboxylate interacts with a helix E arginine, R692 (Figure 2C), then the γ-carboxylate forms interactions with R518 (Figure 2D). The GluN2A LBD helix E is homologous to helix F on both the GluN1 and GluA2 LBDs. Protein-ligand contacts at the α- and γ-carboxylates are broken, and the ligand flips twice, trading interaction partners between the α- and γ-carboxylates (Figures 2D–2I). Next, contacts between helix E and glutamate are broken, and glutamate moves into the binding pocket in the inverted orientation while contacting R518 (Figure 2J). Once inside the binding pocket, the glutamate ligand contacts residues on lobe 1 prior to interacting with residues on lobe 2. The establishment of interactions with lobe 1 before lobe 2 is consistent with time-resolved vibrational spectroscopy experiments with the GluA2 LBD (Cheng et al., 2005Cheng Q. Du M. Ramanoudjame G. Jayaraman V. Evolution of glutamate interactions during binding to a glutamate receptor.Nat. Chem. Biol. 2005; 1: 329-332Crossref PubMed Scopus (35) Google Scholar). Crystal structures of glutamate-bound GluN2A show interactions between the ligand's α-carboxylate and R518, the ligand's amine and S511, T513, and H485, and the ligand's γ-carboxylate with the backbone amines of S689 and T690 (Furukawa et al., 2005Furukawa H. Singh S.K. Mancusso R. Gouaux E. Subunit arrangement and function in NMDA receptors.Nature. 2005; 438: 185-192Crossref PubMed Scopus (603) Google Scholar, Jespersen et al., 2014Jespersen A. Tajima N. Fernandez-Cuervo G. Garnier-Amblard E.C. Furukawa H. Structural insights into competitive antagonism in NMDA receptors.Neuron. 2014; 81: 366-378Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). During our simulations, glutamate forms contacts with R518 alternately at both the α- and γ-carboxylates, flipping between the inverted orientation and the orientation seen in crystal structures. The ligand's amine is also able to interact with S511, T513, and H485, but these interactions are weaker in the inverted conformation. Vibrational spectroscopic studies with the GluA2 LBD indicate that partial agonists have weaker interactions at the α-amine than full agonists (Cheng and Jayaraman, 2004Cheng Q. Jayaraman V. Chemistry and conformation of the ligand-binding domain of GluR2 subtype of glutamate receptors.J. Biol. Chem. 2004; 279: 26346-26350Crossref PubMed Scopus (19) Google Scholar, Mankiewicz et al., 2007Mankiewicz K.A. Rambhadran A. Du M. Ramanoudjame G. Jayaraman V. Role of the chemical interactions of the agonist in controlling alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor activation.Biochemistry. 2007; 46: 1343-1349Crossref PubMed Scopus (17) Google Scholar), which suggests that the inverted ligand conformation in GluN2A may elicit receptor behavior consistent with a partial agonist. In our trajectories, lobe 2 interactions with the ligand are only made between S689 and the α-carboxylate, while the γ-carboxylate contacts R518, resulting in an inverted conformation for the ligand and incomplete cleft closure. It is possible, though, that additional sampling in this state could lead to glutamate rotating to the orientation seen in crystal structures, consequently permitting full cleft closure, as observed in glycine binding to the GluN1 LBD (see below). This inverted bound conformation has been seen in glutamate binding to the GluA2 LBD, although the potential physiological relevance of this binding mode is currently unknown (Yu et al., 2018Yu A. Salazar H. Plested A.J.R. Lau A.Y. Neurotransmitter funneling optimizes glutamate receptor kinetics.Neuron. 2018; 97: 139-149.e4Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). For several trajectories in which the α-carboxylate contacts R518 (T1–5), the γ-carboxylate does not form the requisite lobe 2 contacts for stable binding, and the ligand subsequently dissociates. Since H485 contacts the glutamate ligand in crystal structures (Furukawa et al., 2005Furukawa H. Singh S.K. Mancusso R. Gouaux E. Subunit arrangement and function in NMDA receptors.Nature. 2005; 438: 185-192Crossref PubMed Scopus (603) Google Scholar, Jespersen et al., 2014Jespersen A. Tajima N. Fernandez-Cuervo G. Garnier-Amblard E.C. Furukawa H. Structural insights into competitive antagonism in NMDA receptors.Neuron. 2014; 81: 366-378Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), it is possible that different protonation states of this residue might affect the ligand binding process. We therefore performed ligand-binding simulations in which either the ε-nitrogen or δ-nitrogen were protonated. The resulting pathways turned out to not be significantly different. In GluN1, the analogous residue that coordinates the glycine ligand is F484. Our atomistic simulations of glycine binding to the GluN1 LBD consist of five trajectories that total 25.1 μs. In contrast to glutamate binding into the GluN2A or GluA2 LBDs, glycine binding into the GluN1 LBD is not characterized by interactions with residues outside of the binding pocket. The homologous helix F arginines, R694 and R695, do not interact significantly with the ligand prior to entry into the pocket. In trajectory T6 (Video S2, Table 1), glycine initially enters the cleft via the ξ1 side of the LBD and quickly docks to binding pocket residues on lobe 1 of the LBD (Figures 3A and 3B ). Protein-ligand interactions form between the open LBD and the ligand, first between R523 and the ligand's α-carboxylate (Figure 3C) and then between F484 and the glycine carbons (Figures 3D and 3E). Notably, F484 rotates ∼108° around its χ1 dihedral angle prior to recruiting the ligand (Figures 3B and 3C). Next, the ligand rotates so that its amine contacts the P516 carbonyl oxygen and the hydroxyl group of the T518 sidechain (Figures 3E and 3F). Once glycine forms these contacts, the ligand is in the same conformation seen in crystal structures (Furukawa and Gouaux, 2003Furukawa H. Gouaux E. Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core.EMBO J. 2003; 22: 2873-2885Crossref PubMed Scopus (398) Google Scholar, Furukawa et al., 2005Furukawa H. Singh S.K. Mancusso R. Gouaux E. Subunit arrangement and function in NMDA receptors.Nature. 2005; 438: 185-192Crossref PubMed Scopus (603) Google Scholar, Jespersen et al., 2014Jespersen A. Tajima N. Fernandez-Cuervo G. Garnier-Amblard E.C. Furukawa H. Structural insights into competitive antagonism in NMDA receptors.Neuron. 2014; 81: 366-378Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar); although at this stage, if the LBD does not immediately close, either water molecules in bulk solvent or free glycine ligands can displace the bound glycine from lobe 1. After the LBD closes, we do not observe dissociation of glycine from the ligand-bound, closed conformation.Table 1Trajectories of the Simulation SystemsTrajectorySystemSimulation Time (μs)T1GluN2A LBD, 10 glutamates (H485, prot-εN)5T2GluN2A LBD, 10 glutamates (H485, prot-εN)5T3GluN2A LBD, 10 glutamates (H485, prot-δN)6.6T4GluN2A LBD, 10 glutamates (H485, prot-δN)4T5GluN2A LBD, 10 glutamates (H485, prot-δN)2.859T6GluN1 LBD, 10 glycines2T7GluN1 LBD, 10 glycines10T8GluN1 LBD, 10 glycines2T9GluN1 LBD, 1 glycine5.538T10GluN1 LBD, 1 D-serine5.549T11GluN2A LBD, 1 glutamate1.492T12GluN2A LBD, 1 glutamate1.492T13GluN2A LBD, 1 glutamate4.205T14GluN2A LBD, 1 glutamate1.497T15GluN2A LBD, 1 glutamate2.826T16GluN1/GluN2A LBD dimer, 10 glycines, 10 glutamates5.166T17GluN1/GluN2A LBD dimer, 9 glycines, 10 glutamates5.344T18GluN1/GluN2A LBD dimer, 1 glycine, 1 glutamate4.171total74.739 Open table in a new tab The ligand's amine is more mobile than the glycine carboxylate, once attached to lobe 1 residues. In several instances, contacts between the P516 carbonyl with the ligand's amine and T518 with the ligand's amine are broken, and the amine flips toward lobe 2 of the LBD to briefly interact with S688 and D732, causing the LBD to partially close to (ξ1,ξ2)=(10.8Å,12.7Å) (Figures 3G and 3H). Large conformational change occurs once the ligand's amine stably contacts the D732 sidechain carboxylate, and the LBD closes to (ξ1,ξ2)=(9.2Å,10.3Å) (Figures 3I and 3J). Further interdomain interactions are formed across the two lobes of the binding cleft that lock the LBD closed and contribute to the stability of the closed cleft conformation (Video S3). Helix F residues R694 and R695 on lobe 2 contact E488 and E522 on lobe 1 at the ξ1 side of the binding pocket. Cross-lobe interactions also form between K483 and E712, and N499/G485 and Q686, as seen in crystal structures of partial agonists bound to GluN1 (Inanobe et al., 2005Inanobe A. Furukawa H. Gouaux E. Mechanism of partial agonist action at the NR1 subunit of NMDA receptors.Neuron. 2005; 47: 71-84Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), although these interactions are more transient, and break and reform throughout the simulation. After the LBD closes around the ligand, conformational change relative to the two lobes of the LBD is still possible. Helix H shifts upward toward lobe 1 when R722 contacts E406, and helix F shifts either closer to or away from lobe 1 on the basis of the strength of the following interactions: R694 with E488 and R695 with E522. The formation and breakage of these interdomain interactions is likely the molecular basis for dynamical motions detected in prior simulations (Yao et al., 2013Yao Y. Belcher J. Berger A.J. Mayer M.L. Lau A.Y. Conformational analysis of NMDA receptor GluN1, GluN2, and GluN3 ligand-binding domains reveals subtype-specific characteristics.Structure. 2013; 21: 1788-1799Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and suggest conformational changes can occur in iGluR LBDs beyond simple cleft closure. The cross-lobe interactions positioned at helix F, including R694-E488 and R695-E522, and at helix H, including R722-E406, are more stable during the simulations than K483-E712 and N499/G485-Q686, identified by Inanobe et al., 2005Inanobe A. Furukawa H. Gouaux E. Mechanism of partial agonist action at the NR1 subunit of NMDA receptors.Neuron. 2005; 47: 71-84Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar. We calculated the three-dimensional free energy landscape, or potential of mean force (PMF), for glutamate binding to the GluN2A LBD and glycine binding to the GluN1 LBD. The PMF for GluN2A features several binding pathways and multiple metastable minima (Figures 4A–4C ). The global free energy minimum contains three separate states of the ligand, which largely reflect interactions between the glutamate's α- or γ-carboxylate and R518 (Figure 4A). In state 1, the carboxylate that does not interact with R518 forms contacts within the binding pocket, whereas in states 2 and 3, this carboxylate is stabilized by contacts with arginines on helix E, R692 and R695. At intermediate energetic contours (1.70 kcal/mol), significant ligand density is positioned in the cavity between helices C and E, suggesting that ligand entry to the binding pocket occurs on the ξ1 side of the binding cleft (Figure 4B). The PMF contoured to 2.15 kcal/mol shows continuous density from the surface of the LBD into the binding pocket, indicative of at least three binding pathways, two of which may involve metastable interactions between the ligand's carboxylates and helix E arginines, R692 and R695, and R518 (Figure 4C). Glutamate binding to the GluN2A LBD can therefore be described as a “guided-diffusion” process, where binding assistance is provided by surface-exposed arginines. Error analysis is presented in Figure 5.Figure 5The PMF for Glutamate Binding to the GluN2A LBD and Associated Error AnalysisShow full caption(A–F) The free energy landscape is contoured at (A) 0.6 kcal/mol, (B) 1.2 kcal/mol, (C) 1.8 kcal/mol, (D) 2.4 kcal/mol, (E) 3.0 kcal/mol, and (F) 3.6 kcal/mol.(G–L) The standard deviation of the PMF is contoured at (G) 0.9 kcal/mol, (H) 1.05 kcal/mol, (I) 1.20 kcal/mol, (J) 1.35 kcal/mol, (K) 1.50 kcal/mol, and (L) 1.65 kcal/mol. Ligand positions at the protein surface and in bulk solvent (G) have low statistical uncertainty, with standard deviations of <0.9 kcal/mol. Ligand positions within the binding cleft (I–L) have higher statistical uncertainty, with standard deviations ranging from 1.20 kcal/mol to 1.65 kcal/mol. Statistical uncertainty was calculated using the approach of block averaging.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A–F) The free energy landscape is contoured at (A) 0.6 kcal/mol, (B) 1.2 kcal/mol, (C) 1.8 kcal/mol, (D) 2.4 kcal/mol, (E) 3.0 kcal/mol, and (F) 3.6 kcal/mol. (G–L) The standard deviation of the PMF is contoured at (G) 0.9 kcal/mol, (H) 1.05 kcal/mol, (I) 1.20 kcal/mol, (J) 1.35 kcal/mol, (K) 1.50 kcal/mol, and (L) 1.65 kcal/mol. Ligand positions at the protein surface and in bulk solvent (G) have low statistical uncertainty, with standard deviations of <0.9 kcal/mol. Ligand positions within the binding cleft (I–L) have higher stati" @default.
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• W2806876216 title "Glutamate and Glycine Binding to the NMDA Receptor" @default.
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