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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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Summary of Xenopus laevis NMDA crystallization constructsa, b, Cartoon representation of amino terminal domain (ATD), ligand binding domain (LBD) and transmembrane domain (TMD) for  (a) GluN1 Δ2 and  (b) GluN2B Δ2 subunit constructs. Location of point mutations are highlighted in white circles. Location of deletions are highlighted with a yellow wedge. Mutated glycosylation sites are not shown and are listed in Extended Data Table 1. c, d, Select amino acid sequences of constructs used in these studies compared to wildtype sequence to highlight mutations in  (c) GluN1 and  (d) GluN2B. Mutations are numbered and the purpose of each is detailed in Extended Data Table 1.
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Figure 7: Summary of Xenopus laevis NMDA crystallization constructsa, b, Cartoon representation of amino terminal domain (ATD), ligand binding domain (LBD) and transmembrane domain (TMD) for (a) GluN1 Δ2 and (b) GluN2B Δ2 subunit constructs. Location of point mutations are highlighted in white circles. Location of deletions are highlighted with a yellow wedge. Mutated glycosylation sites are not shown and are listed in Extended Data Table 1. c, d, Select amino acid sequences of constructs used in these studies compared to wildtype sequence to highlight mutations in (c) GluN1 and (d) GluN2B. Mutations are numbered and the purpose of each is detailed in Extended Data Table 1.

Mentions: Here we report crystal structures of the GluN1/GluN2B NMDA receptor from Xenopus laevis in complex with the GluN2B-specific allosteric inhibitor, Ro25-698125, the GluN1 and GluN2B partial agonists 1-aminocyclopropane-1-carboxylic acid (ACPC) 26 and trans-1-aminocyclobutane-1,3-dicarboxylic acid (t-ACBD)27, respectively, and the ion channel blocker, MK-801. To enhance the stability of the receptor in detergent micelles and to reduce conformational surface entropy, we replaced the cytoplasmic C-terminus of the GluN1 and GluN2B subunits with 11 residues from the GluA2 C-terminus28 and we introduced a number of mutations into each subunit, ultimately finding a NMDA receptor complex that preserved binding of full and partial agonists and Ro25-6981, together with small but measurable conductance activated by glycine and glutamate, and with channel block by magnesium. To decrease conformational mobility of the extracellular domains, we substituted GluN2B Lys216 to Cys (K216C), resulting in spontaneous disulfide bond formation between GluN2B subunits, improving crystal quality yet reducing agonist-induced ion channel activity (Extended Data Table 1; Extended Data Figs. 1–4). We determined crystal structures of the GluN1/GluN2B K216C receptor at resolutions of 3.7 Å (Structure 1) and 3.9 Å (Structure 2) and refined the structures to reasonable crystallographic residuals and good stereochemistry. In addition, we mapped cation sites in the ATD by exploiting anomalous scattering from a Tb3+ derivative and probed the mobility of the ATD and LBD layers by comparing a non K216C crosslinked structure to the higher resolution K216C structures (Extended Data Table 2; Supplementary Discussion).


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)

Summary of Xenopus laevis NMDA crystallization constructsa, b, Cartoon representation of amino terminal domain (ATD), ligand binding domain (LBD) and transmembrane domain (TMD) for  (a) GluN1 Δ2 and  (b) GluN2B Δ2 subunit constructs. Location of point mutations are highlighted in white circles. Location of deletions are highlighted with a yellow wedge. Mutated glycosylation sites are not shown and are listed in Extended Data Table 1. c, d, Select amino acid sequences of constructs used in these studies compared to wildtype sequence to highlight mutations in  (c) GluN1 and  (d) GluN2B. Mutations are numbered and the purpose of each is detailed in Extended Data Table 1.
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Related In: Results  -  Collection

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Figure 7: Summary of Xenopus laevis NMDA crystallization constructsa, b, Cartoon representation of amino terminal domain (ATD), ligand binding domain (LBD) and transmembrane domain (TMD) for (a) GluN1 Δ2 and (b) GluN2B Δ2 subunit constructs. Location of point mutations are highlighted in white circles. Location of deletions are highlighted with a yellow wedge. Mutated glycosylation sites are not shown and are listed in Extended Data Table 1. c, d, Select amino acid sequences of constructs used in these studies compared to wildtype sequence to highlight mutations in (c) GluN1 and (d) GluN2B. Mutations are numbered and the purpose of each is detailed in Extended Data Table 1.
Mentions: Here we report crystal structures of the GluN1/GluN2B NMDA receptor from Xenopus laevis in complex with the GluN2B-specific allosteric inhibitor, Ro25-698125, the GluN1 and GluN2B partial agonists 1-aminocyclopropane-1-carboxylic acid (ACPC) 26 and trans-1-aminocyclobutane-1,3-dicarboxylic acid (t-ACBD)27, respectively, and the ion channel blocker, MK-801. To enhance the stability of the receptor in detergent micelles and to reduce conformational surface entropy, we replaced the cytoplasmic C-terminus of the GluN1 and GluN2B subunits with 11 residues from the GluA2 C-terminus28 and we introduced a number of mutations into each subunit, ultimately finding a NMDA receptor complex that preserved binding of full and partial agonists and Ro25-6981, together with small but measurable conductance activated by glycine and glutamate, and with channel block by magnesium. To decrease conformational mobility of the extracellular domains, we substituted GluN2B Lys216 to Cys (K216C), resulting in spontaneous disulfide bond formation between GluN2B subunits, improving crystal quality yet reducing agonist-induced ion channel activity (Extended Data Table 1; Extended Data Figs. 1–4). We determined crystal structures of the GluN1/GluN2B K216C receptor at resolutions of 3.7 Å (Structure 1) and 3.9 Å (Structure 2) and refined the structures to reasonable crystallographic residuals and good stereochemistry. In addition, we mapped cation sites in the ATD by exploiting anomalous scattering from a Tb3+ derivative and probed the mobility of the ATD and LBD layers by comparing a non K216C crosslinked structure to the higher resolution K216C structures (Extended Data Table 2; Supplementary Discussion).

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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