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The N-terminal domain of GluR6-subtype glutamate receptor ion channels

Nature Structural & Molecular Biology volume 16, pages 631–638 (2009)Cite this article

Abstract

The amino-terminal domain (ATD) of glutamate receptor ion channels, which controls their selective assembly into AMPA, kainate and NMDA receptor subtypes, is also the site of action of NMDA receptor allosteric modulators. Here we report the crystal structure of the ATD from the kainate receptor GluR6. The ATD forms dimers in solution at micromolar protein concentrations and crystallizes as a dimer. Unexpectedly, each subunit adopts an intermediate extent of domain closure compared to the apo and ligand-bound complexes of LIVBP and G protein–coupled glutamate receptors (mGluRs), and the dimer assembly has a markedly different conformation from that found in mGluRs. This conformation is stabilized by contacts between large hydrophobic patches in the R2 domain that are absent in NMDA receptors, suggesting that the ATDs of individual glutamate receptor ion channels have evolved into functionally distinct families.

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Figure 1: Expression and purification of the GluR6 ATD.
Figure 2: The GluR6 ATD forms dimers with micromolar affinity.
Figure 3: The GluR6 ATP crystallizes as a dimer.
Figure 4: The GluR6 ATD and mGluR dimers have different conformations.
Figure 5: Dimer interactions in domain R1.
Figure 6: The R2 domain has a hydrophobic patch that is absent in NMDA receptors.

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Acknowledgements

We thank E. Gouaux for sharing GluR2 ATD coordinates; C. Glasser and A. Balbo for technical assistance; D. Leahy, A. Plested and C. Chaudhry for advice and discussion; P. Seeburg (Max-Planck Institute, Heidelberg) and S. Heinemann (Salk Institute) for the gift of the wild-type iGluR plasmids; S. Hansen and P. Reeves (Massachusetts Institute of Technology) for the gift of GnTI− cells; H. Jaffe (Protein/Peptide Sequencing Facility, US National Institute of Neurological Disorders and Stroke (NINDS)) for performing MS analysis and N-terminal sequencing; T. Kawate and E. Gouaux (Vollum Institute) for the gift of Endo H and PNGase F clones; P. Paoletti for providing coordinates for NR2A and NR2B ATD models; and J. Garcia de la Torre (University of Murcia) for the program HYDROPRO. Nucleic acid sequencing was performed by the NINDS DNA sequencing facility. Synchrotron diffraction data was collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. This work was supported by the intramural research program of the US National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services (M.L.M.).

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Authors and Affiliations

  1. Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, National Institute of Child Health and Human Development, Bethesda, Maryland, USA

    Janesh Kumar & Mark L Mayer

  2. Department of Health and Human Services (DHHS), Dynamics of Macromolecular Assembly, Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and BioEngineering, National Institutes of Health (NIH), Bethesda, Maryland, USA

    Peter Schuck

  3. Burnham Institute for Medical Research, La Jolla, California, USA

    Rongsheng Jin

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Contributions

J.K. performed biochemistry and structural biology experiments; P.S. performed analytical ultracentrifugation; M.L.M. assisted with data collection, structure solution and analysis; R.J. solved the GluR2 structure used for molecular replacement; all authors contributed to data analysis and interpretation.

Corresponding author

Correspondence to Mark L Mayer.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Methods and Supplementary Results (PDF 20697 kb)

Supplementary Movie 1

The initial view shows a GluR6 ATD dimer with the N-terminus facing the viewer and the molecular surface of the two protomers shaded blue and red; following rotation by 90° to show a side view, the movie pauses to show the 20° tilt of each subunit about the vertical axis. Next, the surface is colored by electrostatic potential contoured ± 5 kT/e calculated with APBS, and the view rotated by 180° to illustrate the rough solvent exposed polar surface of the GluR6 ATD dimer. The rear subunit is then removed and the view rotated by 180° to illustrate the smooth and non polar surface of the dimer interface in domains R1 and especially R2. The view then rotates to show the lateral edge of domain R1 which contains a cation binding site with a bound cation drawn as a yellow sphere. (MOV 4480 kb)

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Kumar, J., Schuck, P., Jin, R. et al. The N-terminal domain of GluR6-subtype glutamate receptor ion channels. Nat Struct Mol Biol 16, 631–638 (2009). https://doi.org/10.1038/nsmb.1613

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