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References listed in PDB file
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Key reference
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Title
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Distinct structural features of cyclothiazide are responsible for effects on peak current amplitude and desensitization kinetics at iglur2.
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Authors
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H.Hald,
P.K.Ahring,
D.B.Timmermann,
T.Liljefors,
M.Gajhede,
J.S.Kastrup.
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Ref.
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J Mol Biol, 2009,
391,
906-917.
[DOI no: ]
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PubMed id
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Abstract
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Ionotropic glutamate receptors (iGluRs) mediate fast excitatory
neurotransmission. Upon glutamate application,
2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptors undergo
rapid and almost complete desensitization that can be attenuated by positive
allosteric modulators. The molecular mechanism of positive allosteric modulation
has been elucidated previously by crystal structures of the ligand-binding core
of iGluR2 in complex with, for example, cyclothiazide (CTZ). Here, we
investigate the structure and function of CTZ and three closely related
analogues NS1493, NS5206, and NS5217 at iGluR2, by X-ray crystallography and
fast application patch-clamp electrophysiology. CTZ was the most efficacious and
potent modulator of the four compounds on iGluR2(Q)(i) [E(max) normalized to
response of glutamate: 754% (CTZ), 490% (NS1493), 399% (NS5206), and 476%
(NS5217) and EC(50) in micromolar: 10 (CTZ), 26 (NS1493), 43 (NS5206), and 48
(NS5217)]. The four modulators divide into three groups according to efficacy
and desensitization kinetics: (1) CTZ increases the peak current efficacy twice
as much as the three analogues and nearly completely blocks receptor
desensitization; (2) NS5206 and NS5217 have low efficacy and only attenuate
desensitization partially; (3) NS1493 has low efficacy but nearly completely
blocks receptor desensitization. A hydrophobic substituent at the 3-position of
the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system is important
for compound efficacy, with the following ranking: norbornenyl
(bicyclo[2.2.1]hept-2-ene)>cyclopentyl>methyl. The replacement of the
norbornenyl moiety with a significantly less hydrophobic cyclopentane ring
increases the flexibility of the modulator as the cyclopentane ring adopts
various conformations at the iGluR2 allosteric binding site. The main structural
feature responsible for a nearly complete block of desensitization is the
presence of an NH hydrogen bond donor in the 4-position of the
1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system, forming an
anchoring hydrogen bond to Ser754. Therefore, the atom at the 4-position of CTZ
seems to be a major determinant of receptor desensitization kinetics.
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Figure 1.
Fig. 1. Chemical structures of CTZ, NS1493, NS5206, and
NS5217. The atom numbering for relevant atoms is shown in
italics. NS1493, NS5206, and NS5217 are shown in neutral forms.
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Figure 4.
Fig. 4. Binding modes of the allosteric modulators (a) CTZ,
(b) NS1493, (c) NS5206, and (d) NS5217 in iGluR2 S1S2J-N754S. An
F[o] − F[c] omit electron density map (green; contoured at 3
σ), a final 2F[o] − F[c] electron density map (light blue;
contoured at 1 σ), and potential hydrogen-bonding interactions
within 3.2 Å (broken lines) are shown. Oxygen atoms are
colored red, nitrogen atoms are colored blue, and sulfur atoms
are colored yellow. Water molecules are shown as red spheres.
The chemical structures of the compounds are shown in Fig. 1.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2009,
391,
906-917)
copyright 2009.
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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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Mechanism of glutamate receptor desensitization.
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Authors
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Y.Sun,
R.Olson,
M.Horning,
N.Armstrong,
M.Mayer,
E.Gouaux.
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Ref.
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Nature, 2002,
417,
245-253.
[DOI no: ]
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PubMed id
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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.
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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.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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