doi: 10.1126/science.aaw8250. Epub 2019 Apr 11.
Architecture and subunit arrangement of native AMPA receptors elucidated by cryo-EM
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- PMID:30975770
- PMCID: PMC6701862
- DOI: 10.1126/science.aaw8250
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Architecture and subunit arrangement of native AMPA receptors elucidated by cryo-EM
Yan Zhao et al. Science..
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Abstract
Glutamate-gated AMPA receptors mediate the fast component of excitatory signal transduction at chemical synapses throughout all regions of the mammalian brain. AMPA receptors are tetrameric assemblies composed of four subunits, GluA1-GluA4. Despite decades of study, the subunit composition, subunit arrangement, and molecular structure of native AMPA receptors remain unknown. Here we elucidate the structures of 10 distinct native AMPA receptor complexes by single-particle cryo-electron microscopy (cryo-EM). We find that receptor subunits are arranged nonstochastically, with the GluA2 subunit preferentially occupying the B and D positions of the tetramer and with triheteromeric assemblies comprising a major population of native AMPA receptors. Cryo-EM maps define the structure for S2-M4 linkers between the ligand-binding and transmembrane domains, suggesting how neurotransmitter binding is coupled to ion channel gating.
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
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Figures
(A) Flow chart for nAMPAR isolation.(B) Representative SEC profile of the nAMPAR complex. Inset shows a SDS-PAGE of a nAMPAR sample used for cryo-EM grid preparation and of antibody fragments, visualized by Coomassie blue staining.(C) Western blot analysis of isolated nAMPAR blotted using antibodies against GluA1, GluA2, GluA3, GluA4 or PSD-95.(D) Single point SPA radio-ligand binding assay of3H-AMPA binding to the nAMPAR complex. Non specific binding was estimated by the addition of 10 mM cold glutamate (right column). Data are shown as means ± SEM (n=3).(E) nAMPAR subunit composition determination by LC-SRM. Each point represents the peptide-level quantitation shown as percent composition of the original sample. Analysis shows both the median with standard deviation (box plot) and the mean (diamond plot) for each protein subunit after protein-level roll-up.
(A-J) Cryo EM maps of the 10 resolved complexes, viewed parallel to the membrane. Three rules were applied to define subunit position: a) When GluA2 is present at the A/C position, we prioritize A; b) GluA1 is preferentially assigned to the A position; c) GluA3 is preferentially assigned to the C position. GluA1, GluA2, GluA3 and unidentified subunits are blue, red, orange and grey, respectively. The 11B8 scFv, 15F1 Fab and 5B2 Fab are light blue, pink and light cyan, respectively. A schematic cartoon in each panel shows the subunit arrangement of the ATD layer and of antibody fragment binding.(K) Complex distribution illustrated as a pie chart.(L) A pie chart, organized like the ATD layer, showing how the GluA1, GluA2, GluA3 and non-Fab/scFv bound subunit populate the A, B, C, or D positions. Assignment of each subunit position is derived from the view of nAMPAR shown in panel A-J.
(A) Structure of the nAMPAR A3A2A3A2 complex.N-linked carbohydrates are shown as ‘sticks’.(B) ATD, LBD and TMD layers viewed from the ‘top’ of the receptor, along the overall 2-fold axis of symmetry, showing the ATD, LBD and TMD layers.(C-D) Cartoon representations of the A-C and B-D subunits of the nAMPAR A3A2A3A2 complex (C) or the cross-linked A2A3A2A3 complex (27) (D), viewed parallel to the membrane. COMs of R1 and R2 lobes are indicated by black dots. The separations of the R1 or R2 lobes from opposing subunits are labeled at the top of panels. The COM distances between T625 and the entire ATD layer is indicated on the left of the panel. GluA2 and GluA3 subunits of the recombinant cross-linked receptor are red and blue, respectively.
(A-C) Capital ‘Y’ view of di-heteromeric A1A2A1A2 (A), A3A2A3A2 (B) or tri-heteromeric A1A2A3A2 complexes (C). The COM distances between the ATD and LBD layers, and between the LBD and TMD layers, are shown. The spaces between the ATD and LBD layers are indicated by a square bracket. A’-C’ and B’-D’ auxiliary proteins are grey and green.(D) ATD layer analysis of the A1A2A1A2 and A3A2A3A2 complexes. The ATD model of the A1A2A1A2 complex is in the left panel, in which the COMs of the R1 and R2 lobes are indicated by black dots. The distances and angles of vectors defined by COMs of the A1A2A1A2 (upper right) or the A3A2A3A2 (lower right) complex are shown in the right two panels.(E) Interactions that may stabilize the ATD dimer-dimer interface. ‘Top’ view of the ATD layer of the A3A2A3A2 complex is in the left panel. Boxed regions in the right panels highlight dimer-dimer contacts, viewed parallel or perpendicular to the overall 2-fold axis. The α7 helix is colored from blue at the N-terminus to red at the C-terminus. His 208 andN-linked carbohydrates are in ‘sticks’.(F-G) ATD-LBD interface comparison between GluA1 and GluA3 subunits from A1A2A1A2 and A3A2A3A2 complexes or from the A1A2A3A2 complex. Structures were superimposed using the LBD layer. COMs of the D1 lobe, GluA1 R1/R2 lobes and GluA3 R1/R2 lobes are indicated by black, blue and orange dots. Distances are in ängstroms (Å).
(A-C) LBD-TMD structure from opposing subunit pairs, A-C (A) and B-D (B), and adjacent subunits A-D (C), viewed perpendicular to the ‘central’ 2-fold axis (vertical dashed lines). The densities are shown for helices J-K, the S2-M4 linkers and M4 helices. Cαs of the flip/flop splicing sites on the S2-M4 linkers are displayed as black spheres.(D-E) Densities with atomic models for helix K, the S2-M4 linkers from the A/C (D) or the B/D (E) subunits and helix E, the M3-S2 linker from an adjacent subunit, viewed parallel to the membrane. The putative interactions are highlighted by dotted line circles.(F) Density and model of helix K and S2-M4 are shown in the bottom left panel, with the side chains of Y768 and K776.(G) Sequence alignment of GluA1–3 flip and flop isoforms at helix K and the S2-M4 linker. Y768 and G/K776 are highlighted by red and blue boxes, respectively.(H-I) Bar plot showing the effect of Y768A/K776G mutants on desensitization rate and Iss/Ipeak ratio, respectively. An asterisk indicates a statistically significantly difference, p<0.05.
(A) TEVC recordings from oocytes injected with proteoliposomes containing the isolated nAMPAR complex. A representative pair of currents recorded using the same oocyte is shown in the left panel. The right panel shows that the ratio of steady-state currents evoked by kainate (KA) and glutamate (Glu) is 0.50±4 (mean ± standard deviation, n=4).(B) Western blot of nAMPAR used for cryo-EM study with antibodies against TARP γ2, TARP γ8 and cornichon2/3.(C) The LBD-TMDmerged map illustrates architecture of the nAMPAR complex with auxiliary proteins. Auxiliary proteins at the A’/C’ and B’/D’ positions are in grey and green, respectively.(D) Cross section of cryo-EM maps at the ‘height’ indicated in panel C shows the auxiliary protein density features in the A1A2A1A2, A3A2A3A2 and A1A2A3A2 complexes. The auxiliary density features are separated from the receptor TMD by dotted lines.(E) Density with atomic model for B’/D’ auxiliary proteins, viewed parallel to the membrane.(F) Superimposition of auxiliary protein structures from the A’/C’ and B’/D’ positions using related receptor TMDs, viewed from extracellular (upper) or intracellular sides (lower). The relative rotation angles of TM1, TM2 and TM4 are indicated.
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