Conformational Analysis of NMDA Receptor GluN1, GluN2, and GluN3 Ligand-Binding Domains Reveals Subtype-Specific Characteristics

Yao, Yongneng; Belcher, John D.; Berger, Anthony J.; Mayer, Mark L.; Lau, Albert Y.

doi:10.1016/j.str.2013.07.011

Cited by 87 publications

(172 citation statements)

References 49 publications

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“…This distance is comparable with the 41 Å distance measured in the apo crystal structure. The fact that the different liganded states show occupancy covering this entire range suggests that the protein probes both the closed-and open cleft-conformational states (24). The small fraction of occupancies (less than 10%) at efficiencies below 0.6 reflect hyperextended open-cleft conformations that are most likely accessible due to the isolated nature of the agonist-binding domain and the absence of the membrane and amino-terminal domain.…”

Section: Cysteine Labeling Versus Unnatural Amino Acid Labeling-mentioning

confidence: 99%

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Structural Dynamics of the Glycine-binding Domain of the N-Methyl-d-Aspartate Receptor

Dolino¹,

Cooper

Ramaswamy³

et al. 2015

Journal of Biological Chemistry

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Background:The agonist glycine binds to a cleft in the bilobed extracellular domain of NMDA receptors. Results: The full agonist-bound forms of the agonist-binding domain populate more of the higher efficiency closed-cleft states of the receptor. Conclusion: Cleft closure dynamics differ for the full and partial agonist-bound forms. Significance: Dynamics and the extent of cleft closure control agonist efficacy.

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Section: Cysteine Labeling Versus Unnatural Amino Acid Labeling-mentioning

confidence: 99%

“…1 To whom correspondence may be addressed: Dept. (24). Although the theoretical studies shed light on the apo-and glycine-bound states of the NMDA receptor, the mechanism of partial agonism at the agonist-binding domain is still largely unknown.…”

mentioning

confidence: 99%

Structural Dynamics of the Glycine-binding Domain of the N-Methyl-d-Aspartate Receptor

Dolino¹,

Cooper

Ramaswamy³

et al. 2015

Journal of Biological Chemistry

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“…The structures of isolated LBDs from various iGluR subunits have been determined by x-ray crystallography in an assortment of ligand-bound forms (2,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). For AMPA receptor LBDs, the degree of agonist-induced cleft closure is correlated with agonist efficacy (14,15,24).…”

Section: Introductionmentioning

confidence: 99%

“…The free-energy landscapes of LBD cleft opening/closure were explored in molecular-dynamics free-energy simulations (21,(33)(34)(35). Notably, the free-energy landscapes of GluN1 LBDs bound with agonists spanning a range of efficacies showed similar positions for the cleft-closed basins, but the broadness of the basins increased with decreasing agonist efficacy (35).…”

Section: Introductionmentioning

confidence: 99%

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

Dai

Zhou

2016

Biophysical Journal

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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.

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“…To obtain the structure of the GluN1a LBD in the complex with an antagonist, Kvist et al (2013) purified the LBD in the presence of glycine and then dialyzed against a glycine-free buffer before incubating with the antagonist at saturation. To obtain the structure of the unbound state, it was necessary to add the lower affinity ligand l-serine to all chromatography buffers during purification and to add an extensive dialysis step (volume change of 10 15 over 3-4 d) prior to crystallization setup (Yao et al, 2013).…”

Section: Co-crystallizationmentioning

confidence: 99%

Guidelines for the successful generation of protein–ligand complex crystals

Müller

2017

Acta Crystallogr D Struct Biol

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With continuous technical improvements at synchrotron facilities, datacollection rates have increased dramatically. This makes it possible to collect diffraction data for hundreds of protein-ligand complexes within a day, provided that a suitable crystal system is at hand. However, developing a suitable crystal system can prove challenging, exceeding the timescale of data collection by several orders of magnitude. Firstly, a useful crystallization construct of the protein of interest needs to be chosen and its expression and purification optimized, before screening for suitable crystallization and soaking conditions can start. This article reviews recent publications analysing large data sets of crystallization trials, with the aim of identifying factors that do or do not make a good crystallization construct, and gives guidance in the design of an expression construct. It provides an overview of common protein-expression systems, addresses how ligand binding can be both help and hindrance for protein purification, and describes ligand co-crystallization and soaking, with an emphasis on troubleshooting.

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Conformational Analysis of NMDA Receptor GluN1, GluN2, and GluN3 Ligand-Binding Domains Reveals Subtype-Specific Characteristics

Cited by 87 publications

References 49 publications

Structural Dynamics of the Glycine-binding Domain of the N-Methyl-d-Aspartate Receptor

Structural Dynamics of the Glycine-binding Domain of the N-Methyl-d-Aspartate Receptor

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

Guidelines for the successful generation of protein–ligand complex crystals

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