PDBsum entry 1n0t

Membrane protein

PDB id: 1n0t

Contents

Protein chains

257 a.a. *

Ligands

AT1 ×4

SO4 ×4

ACT

Waters ×949

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Competitive antagonism of ampa receptors by ligands of different classes: crystal structure of atpo bound to the glur2 ligand-Binding core, In comparison with dnqx.

Authors

A.Hogner, J.R.Greenwood, T.Liljefors, M.L.Lunn, J.Egebjerg, I.K.Larsen, E.Gouaux, J.S.Kastrup.

Ref.

J Med Chem, 2003, 46, 214-221. [DOI no: 10.1021/jm020989v]

PubMed id

12519060

Abstract

Ionotropic glutamate receptors (iGluRs) constitute a family of ligand-gated ion channels that are essential for mediating fast synaptic transmission in the central nervous system. This study presents a high-resolution X-ray structure of the competitive antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO) in complex with the ligand-binding core of the receptor. Comparison with the only previous structure of the ligand-binding core in complex with an antagonist, 6,7-dinitro-2,3-quinoxalinedione (DNQX) (Armstrong, N.; Gouaux, E. Neuron 2000, 28, 165-181), reveals that ATPO and DNQX stabilize an open form of the ligand-binding core by different sets of interactions. Computational techniques are used to quantify the differences between these two ligands and to map the binding site. The isoxazole moiety of ATPO acts primarily as a spacer, and other scaffolds could potentially be used. Whereas agonists induce substantial domain closures compared to the apo structure, ATPO only induces minor conformational changes. These results are consistent with the hypothesis that domain closure is related to receptor activation. To facilitate the design of novel AMPA receptor antagonists, we present a modified model of the binding site that includes key residues involved in ligand recognition.

Secondary reference #1

Title

Structural basis for ampa receptor activation and ligand selectivity: crystal structures of five agonist complexes with the glur2 ligand-Binding core.

Authors

A.Hogner, J.S.Kastrup, R.Jin, T.Liljefors, M.L.Mayer, J.Egebjerg, I.K.Larsen, E.Gouaux.

Ref.

J Mol Biol, 2002, 322, 93. [DOI no: 10.1016/S0022-2836(02)00650-2]

PubMed id

12215417

Figure 3.
Figure 3. Drawings showing the three agonists and their interactions with the S1S2J protein. (a) 2-Me-Tet-AMPA, (b) ACPA, and (c) Br-HIBO. The bonds of the protein are displayed in yellow and the bound agonists bonds are in blue. Water molecules are shown as red spheres, while remaining atoms are in standard atomic colours (carbon is black, oxygen is red, nitrogen is blue, and bromine is green). Broken lines indicate all potential hydrogen bonds or ionic interactions within 3.3 Å. Radiating spheres indicate hydrophobic contacts within 3.9 Å between carbon atoms in the agonist and neighbouring residues. The only exception is in (c), where hydrophobic contacts between the bromine atom and neighbouring residues are displayed. The binding site of protomer A was employed for (a) and (b), and the binding sites for protomers B and C have similar structures. This Figure was prepared with the program Ligplot.[55.] (d) F[o]−F[c] omit electron density map contoured at 3.0σ for S1S2J:2-Me-Tet-AMPA, S1S2J:ACPA, S1S2J:Br-HIBO, and S1S2J-Y702F:Br-HIBO was prepared by BOBSCRIPT.[56.]

Figure 4.
Figure 4. Surface electrostatic potential of part of the binding site of 2-Me-Tet-AMPA in complex with S1S2J. Positive potential is coloured in blue and negative potential in red, as indicated by the coloured bar to the left. The labelled residues form a well-defined partly hydrophobic and partly polar cavity within the binding site of S1S2J. These residues are within 3.9 Å from the 2-methyltetrazole ring, except from residues Thr686 and Leu704, which are at a distance of 4.2 Å and 4.7 Å, respectively. The ligand 2-Me-Tet-AMPA is shown in ball-and-stick representation, coloured as follows: carbon is white, oxygen is red, and nitrogen is blue. The Figure was prepared with the program Sybyl (Tripos Assoc. Inc.).

The above figures are reproduced from the cited reference with permission from Elsevier

Secondary reference #2

Title

Mechanisms for activation and antagonism of an ampa-Sensitive glutamate receptor: crystal structures of the glur2 ligand binding core.

Authors

N.Armstrong, E.Gouaux.

Ref.

Neuron, 2000, 28, 165-181. [DOI no: 10.1016/S0896-6273(00)00094-5]

PubMed id

11086992

Figure 1.
Figure 1. Ligand Binding Constants for S1S2J(A) Domain structure of iGluRs showing the S1 and S2 segments in turquoise and pink, respectively. “Cut” and “link” denote the edges of the S1S2 construct.(B) K[D] for ^3H-AMPA binding was 24.8 ± 1.8 nM.(C) IC[50] for displacement of ^3H-AMPA by glutamate, kainate, and DNQX were 821 nM, 14.5 μM, and 998 nM, respectively.

Figure 2.
Figure 2. Superposition of the Expanded Cleft Structures and Stereo View of the DNQX Binding Site(A) The two apo molecules (ApoA and ApoB) and two DNQX molecules (DNQXA and DNQXB) in each asymmetric unit were superimposed using only Cα atoms from domain 1. Apo protomers are shaded red and pink while DNQX protomers are colored light green and dark green. DNQX is depicted in black, and selected side chains from DNQXB are shown in dark green. The conformational change undergone by Glu-705 is illustrated by comparing its orientation in ApoB and DNQXB. In the apo state, Glu-705 accepts hydrogen bonds from the side chains of Lys-730 and Thr-655.(B) The chemical structure of DNQX and F[o]-F[c] omit electron density for DNQX and sulfate contoured at 2.5 σ.(C) Stereo image of the interactions between DNQX, sulfate, and S1S2J. DNQXB side chains are colored gray. Water molecules are shown as green balls. DNQX is colored black. Hydrogen bonds between DNQX, sulfate, and S1S2J are indicated by black dashed lines.

The above figures are reproduced from the cited reference with permission from Cell Press

Secondary reference #3

Title

Mechanism of glutamate receptor desensitization.

Authors

Y.Sun, R.Olson, M.Horning, N.Armstrong, M.Mayer, E.Gouaux.

Ref.

Nature, 2002, 417, 245-253. [DOI no: 10.1038/417245a]

PubMed id

12015593

Figure 2.
Figure 2: The L483Y mutation and CTZ stabilize the GluR2 S1S2J dimer. a, Side view of the S1S2J -L483Y dimer in complex with AMPA. Subunit A is grey (domain 1) and blue (domain 2). Subunit B is pink (domain 1) and purple (domain 2). Residues from A are cyan; residues from B are yellow. Lys 505 and Ile 633 flank transmembrane segments 1 and 2, respectively. b, Top view of the L483Y dimer looking down the 2-fold axis. c, CTZ stabilizes the GluR2 S1S2J -N754S dimer by binding in the dimer interface. Side view of the S1S2J dimer in a complex with glutamate and CTZ. The two CTZ molecules are green and are shown in CPK representation. d, Top view of the S1S2J-Glu -CTZ dimer, looking down the 2-fold axis. e, Interactions between Tyr 483 from one subunit and Leu 748 and Lys 752 from another subunit. Similar interactions also occur in the dimer of S1S2J -L483Y in complex with DNQX. Note the intersubunit hydrogen bond between Asn 754 and the carbonyl oxygen of Ser 729. f, Interactions between CTZ and residues from subunits A (cyan) and B (yellow). The black dashed lines are hydrogen bonds and the light blue spheres are water molecules. Stereoviews of e and f are provided in Supplementary Information.

Figure 5.
Figure 5: Agonist-induced conformational changes in the dimer and gating model. a, Overlap of the S1S2J -L483Y dimers bound with either an agonist (AMPA, green) or an antagonist (DNQX, red). The relative movement of the linker region, which connects the ligand-binding core to the channel-forming segments, is represented by the difference in position of Ile 633 in the two structures. Distances between Ile 633 on two protomers are 28.3 � in the DNQX structure and 36.3 � in the AMPA structure. In addition, Ile 633 rotates around the 2-fold axis by 1.25� and moves 2.5 � along the 2-fold axis, away from the membrane. b, A model for glutamate receptor activation and desensitization. Domain 1 and domain 2 of the ligand-binding core are labelled D1 and D2, respectively. Transmembrane segments of each subunit are indicated by a single green cylinder and the N-terminal domain (ATD) has not been included in the model. Each subunit binds a single agonist (A, red circle) and exists in three distinct conformations: closed (C), open (O) and desensitized (D). The closed and open states share the same S1S2 dimer interface. After the binding of agonist, closure of domain 2 towards domain 1 opens the channel gate, whereas closure of domain 1 towards domain 2 disrupts the dimer interface and desensitizes the receptor. The states are connected by using a simplified model for activation and desensitization, more complex versions of which quantitatively describe AMPA receptor responses10,25. A hypothetical plot of the free-energy change occurring during activation and desensitization is shown in the lower left panel for the wild-type (black line), L483Y (green line) and S754D (red line) species.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd

Secondary reference #4

Title

Probing the ligand binding domain of the glur2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct.

Authors

G.Q.Chen, Y.Sun, R.Jin, E.Gouaux.

Ref.

Protein Sci, 1998, 7, 2623-2630. [DOI no: 10.1002/pro.5560071216]

PubMed id

9865957