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NMDA receptor structures reveal subunit arrangement and pore architecture.

Lee CH, Lü W, Michel JC, Goehring A, Du J, Song X, Gouaux E - Nature (2014)

Bottom Line: Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains.The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops.These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

View Article: PubMed Central - PubMed

Affiliation: 1] Vollum Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA [2].

ABSTRACT
N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present X-ray crystal structures of the Xenopus laevis GluN1-GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

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Structural analyses of the transmembrane domain of NMDA receptora, Alpha-carbon superposition of the M3 helices of the GluN1/GluN2B NMDA receptor (Data set 2/Structure 2) onto the corresponding M3 regions of GluA2 receptor (PDB 3KG2; grey). Rmsd is 1.89 Å for 144 aligned α-carbon atoms. The GluN1 subunits are blue and the GluN2B subunits are yellow. b, Amino acid sequence alignment of the NMDA receptor and the KcsA channel in the M2 and M3 regions using Promals3D (http://prodata.swmed.edu/promals3d/promals3d.php). c, Superposition of the four M2 helices of the NMDA receptor onto the corresponding four M2 regions of the KcsA channel (PDB 1K4C; residues 61–75). Rmsd is 1.86 Å. Only chains B and D of the NMDA GluN2B subunits are shown. d, Residual electron density in the central vestibule. Fo-Fc electron density in the central vestibule is shown for the GluN1/GluN2B receptor from Data set 2/Structure 2. For clarity, chain C is removed. e, Fo-Fc electron density map in the central vestibule derived from Data set 1/Structure 1. For clarity, chain B is removed. f, The same electron density map as shown in panel  (e) except that the structure has been rotated by ~90° around the pore axis and chain C of the GluN1 subunit has been removed for clarity. All maps are contoured at 2.8 σ.
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Figure 13: Structural analyses of the transmembrane domain of NMDA receptora, Alpha-carbon superposition of the M3 helices of the GluN1/GluN2B NMDA receptor (Data set 2/Structure 2) onto the corresponding M3 regions of GluA2 receptor (PDB 3KG2; grey). Rmsd is 1.89 Å for 144 aligned α-carbon atoms. The GluN1 subunits are blue and the GluN2B subunits are yellow. b, Amino acid sequence alignment of the NMDA receptor and the KcsA channel in the M2 and M3 regions using Promals3D (http://prodata.swmed.edu/promals3d/promals3d.php). c, Superposition of the four M2 helices of the NMDA receptor onto the corresponding four M2 regions of the KcsA channel (PDB 1K4C; residues 61–75). Rmsd is 1.86 Å. Only chains B and D of the NMDA GluN2B subunits are shown. d, Residual electron density in the central vestibule. Fo-Fc electron density in the central vestibule is shown for the GluN1/GluN2B receptor from Data set 2/Structure 2. For clarity, chain C is removed. e, Fo-Fc electron density map in the central vestibule derived from Data set 1/Structure 1. For clarity, chain B is removed. f, The same electron density map as shown in panel (e) except that the structure has been rotated by ~90° around the pore axis and chain C of the GluN1 subunit has been removed for clarity. All maps are contoured at 2.8 σ.

Mentions: The electron density associated with Data set 2/Structure 2 allowed us to position the polypeptide main chain for the M1-M4 helices of all subunits (Supplementary Video 1). To trace the polypeptide associated with the pore loop, we exploited the continuous electron density for this region in the GluN2B subunit D and, by applying non crystallographic symmetry defined by the transmembrane segments of the other subunits, we traced the three remaining pore loops (Fig. 5a, 5b). The arrangement of transmembrane helices is like that of the GluA2 AMPA receptor28 (Extended Data Fig. 7a), although in the NMDA receptor we have a more complete representation of the ion channel pore and putative selectivity filter. The pre-M1 region of the NMDA receptor forms a ‘collar’ around the extracellular regions of the M3 helices, residing near the boundary of the extracellular side of the membrane. The M1 helix descends across the membrane and makes interactions with the pore-lining M3 helix of the same subunit and the M4 helix of a neighbor. Electron density for the cytoplasmic loop connecting M1 to M2 is weak or missing, and thus this region is absent from the structure. We can visualize the M2 pore helix and most of the extended region of the pore loop forming the selectivity filter and its connection to the N-terminus of M3.


NMDA receptor structures reveal subunit arrangement and pore architecture.

Lee CH, Lü W, Michel JC, Goehring A, Du J, Song X, Gouaux E - Nature (2014)

Structural analyses of the transmembrane domain of NMDA receptora, Alpha-carbon superposition of the M3 helices of the GluN1/GluN2B NMDA receptor (Data set 2/Structure 2) onto the corresponding M3 regions of GluA2 receptor (PDB 3KG2; grey). Rmsd is 1.89 Å for 144 aligned α-carbon atoms. The GluN1 subunits are blue and the GluN2B subunits are yellow. b, Amino acid sequence alignment of the NMDA receptor and the KcsA channel in the M2 and M3 regions using Promals3D (http://prodata.swmed.edu/promals3d/promals3d.php). c, Superposition of the four M2 helices of the NMDA receptor onto the corresponding four M2 regions of the KcsA channel (PDB 1K4C; residues 61–75). Rmsd is 1.86 Å. Only chains B and D of the NMDA GluN2B subunits are shown. d, Residual electron density in the central vestibule. Fo-Fc electron density in the central vestibule is shown for the GluN1/GluN2B receptor from Data set 2/Structure 2. For clarity, chain C is removed. e, Fo-Fc electron density map in the central vestibule derived from Data set 1/Structure 1. For clarity, chain B is removed. f, The same electron density map as shown in panel  (e) except that the structure has been rotated by ~90° around the pore axis and chain C of the GluN1 subunit has been removed for clarity. All maps are contoured at 2.8 σ.
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Related In: Results  -  Collection

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Figure 13: Structural analyses of the transmembrane domain of NMDA receptora, Alpha-carbon superposition of the M3 helices of the GluN1/GluN2B NMDA receptor (Data set 2/Structure 2) onto the corresponding M3 regions of GluA2 receptor (PDB 3KG2; grey). Rmsd is 1.89 Å for 144 aligned α-carbon atoms. The GluN1 subunits are blue and the GluN2B subunits are yellow. b, Amino acid sequence alignment of the NMDA receptor and the KcsA channel in the M2 and M3 regions using Promals3D (http://prodata.swmed.edu/promals3d/promals3d.php). c, Superposition of the four M2 helices of the NMDA receptor onto the corresponding four M2 regions of the KcsA channel (PDB 1K4C; residues 61–75). Rmsd is 1.86 Å. Only chains B and D of the NMDA GluN2B subunits are shown. d, Residual electron density in the central vestibule. Fo-Fc electron density in the central vestibule is shown for the GluN1/GluN2B receptor from Data set 2/Structure 2. For clarity, chain C is removed. e, Fo-Fc electron density map in the central vestibule derived from Data set 1/Structure 1. For clarity, chain B is removed. f, The same electron density map as shown in panel (e) except that the structure has been rotated by ~90° around the pore axis and chain C of the GluN1 subunit has been removed for clarity. All maps are contoured at 2.8 σ.
Mentions: The electron density associated with Data set 2/Structure 2 allowed us to position the polypeptide main chain for the M1-M4 helices of all subunits (Supplementary Video 1). To trace the polypeptide associated with the pore loop, we exploited the continuous electron density for this region in the GluN2B subunit D and, by applying non crystallographic symmetry defined by the transmembrane segments of the other subunits, we traced the three remaining pore loops (Fig. 5a, 5b). The arrangement of transmembrane helices is like that of the GluA2 AMPA receptor28 (Extended Data Fig. 7a), although in the NMDA receptor we have a more complete representation of the ion channel pore and putative selectivity filter. The pre-M1 region of the NMDA receptor forms a ‘collar’ around the extracellular regions of the M3 helices, residing near the boundary of the extracellular side of the membrane. The M1 helix descends across the membrane and makes interactions with the pore-lining M3 helix of the same subunit and the M4 helix of a neighbor. Electron density for the cytoplasmic loop connecting M1 to M2 is weak or missing, and thus this region is absent from the structure. We can visualize the M2 pore helix and most of the extended region of the pore loop forming the selectivity filter and its connection to the N-terminus of M3.

Bottom Line: Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains.The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops.These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

View Article: PubMed Central - PubMed

Affiliation: 1] Vollum Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA [2].

ABSTRACT
N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present X-ray crystal structures of the Xenopus laevis GluN1-GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

Show MeSH