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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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ATD arrangement, cation binding sites and conformational mobilitya, View of the ATD layer along the overall 2-fold axis, from the extracellular side of the membrane, centered on the overall 2-fold axis, and showing the relative location of the underlying LBD layer. Ro25-6981 is green and the K216C disulfide is yellow. The arrangements of subunits for ATD and LBD layers are shown as insets. b, The inverted ATD heterodimeric ‘V’ straddles GluN1 and GluN2B LBD subunits on different local LBD heterodimers. Whereas the ATD R2 lobes interact with the LBDs, the R1 lobes cradle bound Ro25-6981 at an ATD subunit interface. Structure 1 is shown in panels (a) and (b) c, Tb3+ binding sites. Shown is an anomalous difference electron density map, contoured at 3.5 σ (pink mesh). Sites Tb1 and Tb2 are located at the ‘hinge’ between the R1 and R2 lobes whereas sites Tb3 and Tb4 are at receptor-receptor contacts in the crystal lattice. d, Shown are the ATD and LBD extracellular domains derived from the two low resolution GluN1/GluN2B receptor structures (Extended Data Table 2; Data set 4/Structure 4) where the GluN2B subunits do not harbor the K216C disulfide bridge, illustrating the conformational mobility of the ATD layer. The angles between the α5 helices of the GluN2B subunits for each of the two independent receptor complexes in the asymmetric unit illustrate the conformational mobility of the ATD layers.
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Figure 2: ATD arrangement, cation binding sites and conformational mobilitya, View of the ATD layer along the overall 2-fold axis, from the extracellular side of the membrane, centered on the overall 2-fold axis, and showing the relative location of the underlying LBD layer. Ro25-6981 is green and the K216C disulfide is yellow. The arrangements of subunits for ATD and LBD layers are shown as insets. b, The inverted ATD heterodimeric ‘V’ straddles GluN1 and GluN2B LBD subunits on different local LBD heterodimers. Whereas the ATD R2 lobes interact with the LBDs, the R1 lobes cradle bound Ro25-6981 at an ATD subunit interface. Structure 1 is shown in panels (a) and (b) c, Tb3+ binding sites. Shown is an anomalous difference electron density map, contoured at 3.5 σ (pink mesh). Sites Tb1 and Tb2 are located at the ‘hinge’ between the R1 and R2 lobes whereas sites Tb3 and Tb4 are at receptor-receptor contacts in the crystal lattice. d, Shown are the ATD and LBD extracellular domains derived from the two low resolution GluN1/GluN2B receptor structures (Extended Data Table 2; Data set 4/Structure 4) where the GluN2B subunits do not harbor the K216C disulfide bridge, illustrating the conformational mobility of the ATD layer. The angles between the α5 helices of the GluN2B subunits for each of the two independent receptor complexes in the asymmetric unit illustrate the conformational mobility of the ATD layers.

Mentions: Subunit arrangement within the GluN1/GluN2B NMDA receptor adheres to the organization of the AMPA receptor28, with the glycine-binding GluN1 subunits occupying the A/C subunit positions and the glutamate-binding GluN2B subunits situated in the B/D subunit sites (Fig. 2a). In harmony with cross-linking studies on the GluN1/GluN2A receptor28–30 and isolated ATDs31 and in agreement with crystal structures of the GluN1/GluN2A LBDs32 and the GluN1/GluN2B ATDs24, the ATDs and the LBDs are organized as local GluN1/GluN2B heterodimers. Like the AMPA receptor28, there is subunit ‘cross over’ between ATD and LBD layers such that the subunits of a given ATD heterodimer are connected to subunits in a different LBD heterodimer, thus knitting together the extracellular domain superstructure. The TMDs are further stitched together by the M4 helices interacting nearly exclusively with TM segments from an adjacent subunit. The arrangement of subunits within this NMDA receptor complex illustrates how the subunit non equivalence first described for the homomeric AMPA receptor28 has been exploited in an obligatory heteromeric assembly.


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)

ATD arrangement, cation binding sites and conformational mobilitya, View of the ATD layer along the overall 2-fold axis, from the extracellular side of the membrane, centered on the overall 2-fold axis, and showing the relative location of the underlying LBD layer. Ro25-6981 is green and the K216C disulfide is yellow. The arrangements of subunits for ATD and LBD layers are shown as insets. b, The inverted ATD heterodimeric ‘V’ straddles GluN1 and GluN2B LBD subunits on different local LBD heterodimers. Whereas the ATD R2 lobes interact with the LBDs, the R1 lobes cradle bound Ro25-6981 at an ATD subunit interface. Structure 1 is shown in panels (a) and (b) c, Tb3+ binding sites. Shown is an anomalous difference electron density map, contoured at 3.5 σ (pink mesh). Sites Tb1 and Tb2 are located at the ‘hinge’ between the R1 and R2 lobes whereas sites Tb3 and Tb4 are at receptor-receptor contacts in the crystal lattice. d, Shown are the ATD and LBD extracellular domains derived from the two low resolution GluN1/GluN2B receptor structures (Extended Data Table 2; Data set 4/Structure 4) where the GluN2B subunits do not harbor the K216C disulfide bridge, illustrating the conformational mobility of the ATD layer. The angles between the α5 helices of the GluN2B subunits for each of the two independent receptor complexes in the asymmetric unit illustrate the conformational mobility of the ATD layers.
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Related In: Results  -  Collection

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Figure 2: ATD arrangement, cation binding sites and conformational mobilitya, View of the ATD layer along the overall 2-fold axis, from the extracellular side of the membrane, centered on the overall 2-fold axis, and showing the relative location of the underlying LBD layer. Ro25-6981 is green and the K216C disulfide is yellow. The arrangements of subunits for ATD and LBD layers are shown as insets. b, The inverted ATD heterodimeric ‘V’ straddles GluN1 and GluN2B LBD subunits on different local LBD heterodimers. Whereas the ATD R2 lobes interact with the LBDs, the R1 lobes cradle bound Ro25-6981 at an ATD subunit interface. Structure 1 is shown in panels (a) and (b) c, Tb3+ binding sites. Shown is an anomalous difference electron density map, contoured at 3.5 σ (pink mesh). Sites Tb1 and Tb2 are located at the ‘hinge’ between the R1 and R2 lobes whereas sites Tb3 and Tb4 are at receptor-receptor contacts in the crystal lattice. d, Shown are the ATD and LBD extracellular domains derived from the two low resolution GluN1/GluN2B receptor structures (Extended Data Table 2; Data set 4/Structure 4) where the GluN2B subunits do not harbor the K216C disulfide bridge, illustrating the conformational mobility of the ATD layer. The angles between the α5 helices of the GluN2B subunits for each of the two independent receptor complexes in the asymmetric unit illustrate the conformational mobility of the ATD layers.
Mentions: Subunit arrangement within the GluN1/GluN2B NMDA receptor adheres to the organization of the AMPA receptor28, with the glycine-binding GluN1 subunits occupying the A/C subunit positions and the glutamate-binding GluN2B subunits situated in the B/D subunit sites (Fig. 2a). In harmony with cross-linking studies on the GluN1/GluN2A receptor28–30 and isolated ATDs31 and in agreement with crystal structures of the GluN1/GluN2A LBDs32 and the GluN1/GluN2B ATDs24, the ATDs and the LBDs are organized as local GluN1/GluN2B heterodimers. Like the AMPA receptor28, there is subunit ‘cross over’ between ATD and LBD layers such that the subunits of a given ATD heterodimer are connected to subunits in a different LBD heterodimer, thus knitting together the extracellular domain superstructure. The TMDs are further stitched together by the M4 helices interacting nearly exclusively with TM segments from an adjacent subunit. The arrangement of subunits within this NMDA receptor complex illustrates how the subunit non equivalence first described for the homomeric AMPA receptor28 has been exploited in an obligatory heteromeric assembly.

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