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PDBsum entry 2anj
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Membrane protein
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PDB id
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2anj
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References listed in PDB file
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Key reference
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Title
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A binding site tyrosine shapes desensitization kinetics and agonist potency at glur2. A mutagenic, Kinetic, And crystallographic study.
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Authors
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M.M.Holm,
P.Naur,
B.Vestergaard,
M.T.Geballe,
M.Gajhede,
J.S.Kastrup,
S.F.Traynelis,
J.Egebjerg.
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Ref.
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J Biol Chem, 2005,
280,
35469-35476.
[DOI no: ]
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PubMed id
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Abstract
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Binding of an agonist to the
2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid (AMPA) receptor
family of the glutamate receptors (GluRs) results in rapid activation of an ion
channel. Continuous application results in a non-desensitizing response for
agonists like kainate, whereas most other agonists, such as the endogenous
agonist (S)-glutamate, induce desensitization. We demonstrate that a highly
conserved tyrosine, forming a wedge between the agonist and the N-terminal part
of the bi-lobed ligand-binding site, plays a key role in the receptor kinetics
as well as agonist potency and selectivity. The AMPA receptor GluR2, with
mutations in Tyr-450, were expressed in Xenopus laevis oocytes and characterized
in a two-electrode voltage clamp setup. The mutation GluR2(Y450A) renders the
receptor highly kainate selective, and rapid application of kainate to
outside-out patches induced strongly desensitizing currents. When Tyr-450 was
substituted with the larger tryptophan, the (S)-glutamate desensitization is
attenuated with a 10-fold increase in steady-state/peak currents (19% compared
with 1.9% at the wild type). Furthermore, the tryptophan mutant was introduced
into the GluR2-S1S2J ligand binding core construct and co-crystallized with
kainate, and the 2.1-A x-ray structure revealed a slightly more closed ligand
binding core as compared with the wild-type complex. Through genetic
manipulations combined with structural and electrophysiological analysis, we
report that mutations in position 450 invert the potency of two central agonists
while concurrently strongly shaping the agonist efficacy and the desensitization
kinetics of the AMPA receptor GluR2.
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Figure 1.
FIGURE 1. Structure of ionotropic glutamate receptors and
ligands. A, an individual subunit is highlighted on the left and
is formed by a single polypeptide chain beginning with the
N-terminal LIVBP (leucine-isoleucine-valine-binding
protein)-like domain, which is followed by S1 forming the
majority of domain 1 (D1) of the ligand binding core (red). The
amino acid chain then transverses domain 2 (D2) of the ligand
binding core and forms M1, the P-loop, and M2. Subsequently, the
rest of domain 2 (D2, blue) is formed by S2, ending up in the
last transmembrane  -helix (M3) and the
intracellular C-terminal domain. The flip/flop region lies on
the back, primarily of the D1. Scissors indicate where the
corresponding receptor has been cleaved during construction of
the GluR2-S1S2J construct. B, structures of selected AMPA
receptor agonists.
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Figure 7.
FIGURE 7. Details of the structure of GluR2-S1S2J(Y450W) in
complex with kainate. A, schematic representation of the overall
structure of GluR2-S1S2J(Y450W) with zoom on a F[o] - F[c] omit
map contoured at 3.0  for kainate (blue in D1
and green in D2). B, stereo view of the ligand binding pocket of
GluR2-S1S2J(Y450W) showing the hydrogen-bonding network to
kainate (black). C, comparison of the ligand binding pocket of
wild-type GluR2-S1S2J (6) (PDB code 1FT0 [PDB]
, black) and GluR2-S1S2J(Y450W) (blue in D1 and green in D2),
both in complex with kainate. The side chain of residue 450 is
depicted showing the 6° difference of the aromatic wedge.
Nitrogen atoms are blue, oxygen atoms are red, and sulfur atoms
are yellow. The figure was prepared using Molscript (36) and
Raster3d (37).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2005,
280,
35469-35476)
copyright 2005.
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Secondary reference #1
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Title
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Mechanisms for activation and antagonism of an ampa-Sensitive glutamate receptor: crystal structures of the glur2 ligand binding core.
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Authors
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N.Armstrong,
E.Gouaux.
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Ref.
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Neuron, 2000,
28,
165-181.
[DOI no: ]
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PubMed id
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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.
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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.
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The above figures are
reproduced from the cited reference
with permission from Cell Press
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Secondary reference #2
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Title
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Tyr702 is an important determinant of agonist binding and domain closure of the ligand-Binding core of glur2.
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Authors
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A.Frandsen,
D.S.Pickering,
B.Vestergaard,
C.Kasper,
B.B.Nielsen,
J.R.Greenwood,
G.Campiani,
C.Fattorusso,
M.Gajhede,
A.Schousboe,
J.S.Kastrup.
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Ref.
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Mol Pharmacol, 2005,
67,
703-713.
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PubMed id
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Secondary reference #3
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Title
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Tuning activation of the ampa-Sensitive glur2 ion channel by genetic adjustment of agonist-Induced conformational changes.
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Authors
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N.Armstrong,
M.Mayer,
E.Gouaux.
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Ref.
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Proc Natl Acad Sci U S A, 2003,
100,
5736-5741.
[DOI no: ]
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PubMed id
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Figure 4.
Fig. 4. Comparison of WT and S1S2J L650T/AMPA(AS)
conformations. (A) Superposition of WT S1S2J/AMPA (gray) with
S1S2J L650T/AMPA (AS form) protomer A (blue). (B) Superposition
of WT S1S2J/AMPA (gray) with S1S2J L650T/AMPA(AS) protomer B
(green). The black arrows in A and B indicate the axis of
rotation relating the conformational difference between the WT
and L650T structures. (C) Superimposed WT and mutant AMPA dimers.
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Figure 6.
Fig. 6. Mechanism of agonist binding and domain closure.
(A) The binding site of the open-cleft, closed-channel state
(Apo S1S2J, protomer A). (B) The possible first step in agonist
binding as observed in molecule B of the L650T/AMPA(AS)
structure. We suggest that this semiclosed cleft conformation
represents the agonist-bound, closed-channel state. (C) The
closed-cleft, open-channel conformation as observed in the WT
S1S2J/AMPA binding cleft (protomer A). In B and C, water
molecules are shown as green spheres, AMPA is drawn in magenta,
and hydrogen bonds are depicted by black dashed lines. The
degrees of domain closure relative to the Apo conformation are
indicated below each structure.
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