Báo cáo khoa học: Exploring the GluR2 ligand-binding core in complex with the bicyclical AMPA analogue (S)-4-AHCP potx

Exploring the GluR2 ligand-binding core in complex
with the bicyclical AMPA analogue (S)-4-AHCP
Bettina B. Nielsen
1
, Darryl S. Pickering
2
, Jeremy R. Greenwood
1
, Lotte Brehm
1
, Michael Gajhede
1
,
Arne Schousboe
2
and Jette S. Kastrup
1
1 Biostructural Research, Department of Medicinal Chemistry, Danish University of Pharmaceutical Sciences, Copenhagen, Denmark
2 Department of Pharmacology, Danish University of Pharmaceutical Sciences, Copenhagen, Denmark
The main excitatory amino acid in the central nervous
system (S)-glutamate, exerts its actions by binding to
three different classes of ionotropic glutamate receptors
(iGluRs) and three classes of metabotropic receptors,
which all have important functions in neuronal signal-
ling (for a review, see [1]). With the glutamatergic sys-
tem implicated in a variety of brain disorders such as
schizophrenia and Alzheimer’s disease, these receptors
are potential targets for pharmacotherapy [2,3]. The
iGluRs form ligand-gated ion channels and have been
classified according to their agonist selectivity as
2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)pr opionic

acid (AMPA), kainic acid (KA) and N-methyl-d-aspar-
tic acid (NMDA) receptors [3]. Four subunits, assem-
bled as a pair of dimers, constitute the receptor
ion-channel complex [4–11]. Among iGluR subunits,
homomeric and heteromeric receptors constructed from
cloned GluR1–4 are most sensitive to activation by
AMPA, and thus native AMPA receptors are identified
with these genes [12–14]. The subunits exist in two
different alternatively spliced isoforms, flip (i) and flop
(o), which have different desensitization properties.
In addition, two RNA-edited isoforms of GluR2 are
found in which a crucial amino-acid residue in the
channel pore region is either glutamine or arginine
(the Q ⁄ R site) and this affects channel properties, e.g.
rectification and ion-selectivity of GluR2-containing
heteromeric channels [2].
Keywords
(S)-4-AHCP; bicyclical AMPA analogue;
ionotropic glutamate receptor; ligand-binding
core; X-ray crystallography
Correspondence
J. S. Kastrup, Biostructural Research,
Department of Medicinal Chemistry, Danish
University of Pharmaceutical Sciences,
Universitetsparken 2, DK-2100 Copenhagen,
Denmark
Fax: +45 3530 6040
Tel: +45 3530 6486
E-mail:
(Received 28 September 2004, revised 18

January 2005, accepted 25 January 2005)
doi:10.1111/j.1742-4658.2005.04583.x
The X-ray structure of the ionotropic GluR2 ligand-binding core (GluR2-
S1S2J) in complex with the bicyclical AMPA analogue (S)-2-amino-3-(3-hyd-
roxy-7,8-dihydro-6H-cyclohepta[d]-4-isoxazolyl)propionic acid [(S)-4-AHCP]
has been determined, as well as the binding pharmacology of this construct
and of the full-length GluR2 receptor. (S)-4-AHCP binds with a glutamate-
like binding mode and the ligand adopts two different conformations. The K
i
of (S)-4-AHCP at GluR2-S1S2J was determined to be 185 ± 29 nm and at
full-length GluR2(R)
o
it was 175 ± 8 nm.(S)-4-AHCP appears to elicit par-
tial agonism at GluR2 by inducing an intermediate degree of domain closure
(17°). Also, functionally (S)-4-AHCP has an efficacy of 0.38 at GluR2(Q)
i
,
relative to (S)-glutamate. The proximity of bound (S)-4-AHCP to domain
D2 prevents full D1–D2 domain closure, which is limited by steric repulsion,
especially between Leu704 and the ligand.
Abbreviations
4-AHCP, 2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]-4-isoxazolyl)propionic acid; AMPA, 2-amino-3-(3-hydroxy-5-methyl-4-
isoxazolyl)propionic acid; Br-HIBO, 2-amino-3-(4-bromo-3-hydroxy-5-isoxazolyl)propionic acid; EC
50
, concentration of drug producing 50% of
the maximal response; GluR2-S1S2J, soluble construct of the ionotropic GluR2 ligand-binding core; iGluR, ionotropic glutamate receptor; KA,
kainic acid; 2-Me-Tet-AMPA, 2-amino-3-[3-hydroxy-5-(2-methyl-2H-5-tetrazolyl)-4-isoxazolyl]propionic acid; n
H
, Hill coefficient; NMDA,
N-methyl-

D-aspartic acid.
FEBS Journal 272 (2005) 1639–1648 ª 2005 FEBS 1639
Overexpression and purification of a GluR2 construct
(GluR2-S1S2J) containing the extracellular segments S1
and S2 linked by a small peptide has provided a soluble
form of the ligand-binding core of the GluR2 receptor,
belonging to the AMPA class of iGluRs [15]. Binding of
agonists to this construct creates a pharmacological
profile comparable to that seen for the full-length recep-
tor [16,17]. A number of structures of GluR2-S1S2J in
complex with different agonists (e.g. [15,18–22]) and
antagonists [15,23] have provided evidence that compet-
itive ligands bind in a cleft between two domains, D1
and D2. Domain movement occurs upon ligand bind-
ing, resulting in closure of the binding cleft. The extent
of domain closure is correlated with activation and
desensitization of the receptor (for a review, see [24]).
The AMPA receptor agonist, 2-amino-3-(3-hydroxy-
7,8-dihydro-6H-cyclohepta[d] -4-isoxazolyl)propionic
acid (4-AHCP) was originally synthesized as a con-
formationally restricted analogue of AMPA [25]. As is
often the case, the agonist activity resides with one
enantiomer (S)-4-AHCP. The agonist shows activity at
AMPA receptors, but it is 35–115 times more potent
at GluR5 homomers, classified as low affinity KA
receptors [26]. (S)-4-AHCP distinguishes itself from the
majority of iGluR agonists by its bicyclical structure,
and the extra carbon atom between the a-amino acid
moiety and the distal isoxazole 3-hydroxy anion
(Fig. 1). The seven-membered ring offers opportunities

for design and stereospecific derivatization in direc-
tions not easily accessible from other scaffolds. Accu-
rate knowledge of the binding mode of (S)-4-AHCP
may assist in the structure-based design of ligands that
are targeted to iGluR specific subtypes, as well as
highlighting regions that could be occupied by small
hydrophobic substituents to increase affinity. Here, we
present the first structure of GluR2-S1S2J in complex
with the bicyclical AMPA analogue (S)-4-AHCP, as
well as the pharmacology of (S)-4-AHCP binding to
GluR2-S1S2J and full-length GluR2 receptors.
Results and Discussion
Pharmacology of (S)-4-AHCP
The affinity of (S)-4-AHCP for GluR2-S1S2J was deter-
mined to be (mean ± SEM): K
i
¼ 185 ± 29 nm; Hill
coefficient (n
H
) ¼ 1.03 ± 0.04 (n ¼ 4) and for full-
length GluR2(R)
o
:K
i
¼ 175 ± 8 nm;n
H
¼ 0.95 ± 0.02
(n ¼ 3) (Fig. 2A). No statistically significant difference
between the K
i

values was observed using the t-test
(P ¼ 0.80). The affinity for the construct GluR2-S1S2J
is identical to the affinity for the full-length receptor,
implying that the binding of (S)-4-AHCP observed in
the crystal structure represents the binding mode at the
full-length receptor. Analysis of concentration–response
curves for (S)-4-AHCP activation of GluR2(Q)
i
exp-
ressed in Xenopus laevis oocytes gave: concentration of
drug producing 50% of the maximal response (EC
50
) ¼
17.5 ± 1.2 lm,n
H
¼ 0.95 ± 0.04 (n ¼ 7) and an effic-
acy of 0.381 ± 0.046 (n ¼ 7), relative to (S)-glutamate
(Fig. 2B). Similar EC
50
values have been reported for
(S)-4-AHCP at the other AMPA receptors: GluR1
o
(4.5 lm), GluR3
o
(7.2 lm) and GluR4
o
(15 lm) [26].
Interactions of (S)-4-AHCP with GluR2
The GluR2-S1S2J:(S)-4-AHCP complex crystallizes
with one molecule in the asymmetric unit of the crystal

and the structure has been determined at 1.75 A
˚
reso-
lution (Table 1). (S)-4-AHCP was modelled in two dif-
ferent conformations in the ligand-binding site; the
major variation is two conformationally enantiomeric
puckering modes of the seven-membered ring of the
ligand but the orientation of the ligand in the two con-
formations is also slightly different (Fig. 3). However,
the positions of the atoms of the 3-isoxazolol moiety
only differ between 0.2 and 0.4 A
˚
, which may be
within the experimental error. The interactions of the
a-amino acid moiety of both conformations of the lig-
and with binding site residues are tabulated in Table 2.
Multiple ligand conformations, implying some retent-
ion of conformational entropy upon binding, have not
previously been observed at GluR2. The barrier to ring
inversion in (S)-4-AHCP is lower than the barrier to
Fig. 1. Chemical structures of the neutral forms of the GluR2 agon-
ists (S)-glutamate, (S)-AMPA, (S)-2-Me-Tet-AMPA and (S)-4-AHCP
(including atom numbering as in pdb-file).
GluR2 ligand-binding core in complex with (S)-4-AHCP B. B. Nielsen et al.
1640 FEBS Journal 272 (2005) 1639–1648 ª 2005 FEBS
binding and receptor activation, hence, ring inversion
occurs on a much more rapid timescale than binding
and activation. Significant flexibility in the protein is
not seen in response to the two conformations and
both are substantially populated, which suggests that

they are similar in internal energy, in interaction
energy and in binding energy. We expect that the lig-
and can rearrange while bound, at least at the tem-
perature at which affinity is measured. Thus, the
observed affinity will be a Boltzmann average of the
two states in rapid equilibrium.
The interactions of the a-amino acid moiety of the
ligand with binding site residues (Table 2) are con-
served compared to other GluR2-agonist complexes
[15,18–21]. Besides the residues forming ion pairs or
hydrogen bonds with the ligand, a number of addi-
tional residues are involved in hydrophobic or van der
Waals’ interactions (Table 2). Practically all the same
residues have been shown to be in van der Waals’ con-
tact in other agonist complexes (e.g. [20]). The ligand
is ‘squeezed in’ between Tyr450 and Leu650 on the
one side and Glu705 and Tyr732 on the opposite side,
forming a hydrophobic sandwich (Fig. 3B and C). The
Tyr450 side chain interacts extensively with the
a-amino acid group, as well as with C4, C5, C6 and
C7 of the seven-membered ring. Leu650 interacts with
the isoxazole, C4, C5, C6 and C9. The residues
Glu402 and Thr686, which form an interdomain
hydrogen-bond lock upon agonist binding, also inter-
act with atoms of the seven-membered ring. Leu704
forms contacts to C3, N1 and O1 of the isoxazole ring
and C8 of the seven-membered ring, while the residue
Fig. 2. Pharmacology of (S)-AHCP at GluR2. (A) Binding affinities of
(S)-AHCP at the soluble GluR2-S1S2J construct and at the full-
length GluR2(R)

o
receptor. One representative [
3
H]AMPA radiolig-
and binding experiment is shown for each (mean ± SD of tripli-
cates). Experiments were replicated a total of three or four times.
(B) Potency of (S)-AHCP activation of GluR2(Q)
i
expressed in
X. laevis oocytes. Shown are means ± SD of concentration–
response data pooled from seven oocytes, normalized to each
maximal steady-state response. (Inset) Current traces showing the
relative efficacy (here, 0.361) of (S )-4-AHCP vs. (S)-glutamate.
Cyclothiazide (100 l
M CTZ; white box) was preapplied for 60 s
before application of 1 m
M (S)-glutamate (G; black box) or 500 lM
(S)-AHCP (A, black box). Note that a perfusion delay of 4–5 s
occurs in this system. Scale bars, 200 nA, 10 s.
Table 1. Crystal data, data collection and refinement statistics for
GluR2-S1S2J:(S )-4-AHCP.
Crystal data
Space group P2
1
2
1
2
Unit cell parameters (A
˚
)a¼ 94.4, b ¼ 59.5, c ¼ 47.8

No. of molecules per a.u. 1
Data collection
Resolution range (A
˚
)
a
20.1–1.75 (1.78–1.75)
No. of unique reflections 27472
Average redundancy 3.5
Completeness (%) 99.1 (96.3)
R
sym
(%) 3.7 (22.7)
I ⁄ r(I) 26.7 (4.1)
Refinements
Total number of atoms
Non-hydrogen 2393
Protein 2039
Ligand 17
Water 309
Sulfate and glycerol 28
R-values
R
work
(%) 16.9 (24.8)
R
free, 5%
(%) 21.4 (31.8)
Rms deviations
Bond lengths (A

˚
) 0.017
Bond angles (°) 1.6
Residues in allowed regions
of Ramachandran plot (%)
b
99.6
Mean B-values (A
˚
2
)
Protein atoms 18.7
Ligand atoms 14.9
Water 31.5
Sulfate and glycerol 52.5
a
Values in parentheses correspond to the outermost resolution
bin.
b
The Ramachandran plot was calculated according to Kleywegt
and Jones [49].
B. B. Nielsen et al. GluR2 ligand-binding core in complex with (S)-4-AHCP
FEBS Journal 272 (2005) 1639–1648 ª 2005 FEBS 1641
Met708 follows the contour of the seven-membered
ring, interacting hydrophobically with C6, C7 and C8.
(S)-4-AHCP may be classified as a barely exposed lig-
and, as only a small part of the bicyclical ring system
can be seen from the exterior of the protein.
The conformation of the peptide bond Asp651–
Ser652, which has been shown to be flipped 180° in

the complexes with most full agonists [15,18,19], is
similar to the nonflipped conformation observed in,
for example, the apo and (S)-2-amino-3-(3-hydroxy-
5-methyl-4-isoxazolyl)propionic acid [(S)-Br-HIBO]
complex. The backbone oxygen atom of Ser652 is
connected to the 3-hydroxy anion (O2) of (S)-4-AHCP
through the water molecule W1.
Comparison with other isoxazole-based agonists
The binding modes of GluR2 agonists thus far charac-
terized can be broadly divided into two classes, the
glutamate mode and the AMPA mode. In the glutam-
ate binding mode, the agonist approaches D2 more
closely and interacts directly with the hydrogen-bond
donor atoms of Ser654 and Thr655. In the AMPA
binding mode, the interaction with D2 is mediated via
a water molecule (here denoted W4) [15,18,19]. To
characterize the binding mode of (S)-4-AHCP, the
structure was compared to those of GluR2-S1S2J in
complex with (S)-glutamate (S)-AMPA and (S)-2-
amino-3-[3-hydroxy-5-(2-methyl-2 H -5 -tetrazolyl)-4-is-
oxazolyl]propionic acid [(S)-2-Me-Tet-AMPA]. The
binding mode of (S)-4-AHCP falls between those of
(S)-2-Me-Tet-AMPA and (S)-AMPA with regard to
the position of the isoxazole ring (Fig. 4A). As both
(S)-4-AHCP and (S)-2-Me-Tet-AMPA bind with the
distal anionic moiety interacting directly with the back-
bone N atom of Thr655, the binding mode resembles
most closely that of (S)-glutamate. The 3-hydroxy
anion of (S)-4-AHCP is also hydrogen bonded to W1,
as seen for (S)-2-Me-Tet-AMPA and (S)-glutamate,

but no water molecule is observed corresponding to
W4 in the (S)-AMPA complex. The glutamate binding
mode of (S)-4-AHCP would be expected from the size
of the 5-substituent of the isoxazole ring (the fused
seven-membered ring) since the limited hydrophobic
Fig. 3. Binding of (S)-4-AHCP to GluR2-S1-
S2J (shown in stereo). (A) 2F
o
-F
c
electron
density map of (S)-4-AHCP contoured at
1 r. The electron density was generated
with program ARP ⁄ wARP and before the
ligand was introduced into the model. Two
conformations were modelled with confor-
mation 1 of the ligand shown in magenta
and conformation 2 in cyan. Additional F
o
-F
c
electron density (green and contoured at
3 r) was apparent after modelling only con-
formation 1. (B) Selected residues of the
ligand-binding site and their interactions with
the ligand are shown. Dashed lines indicate
hydrogen bonds ⁄ ionic interactions (< 3.3 A
˚
).
The red spheres represent water molecules,

the nitrogen atoms are coloured blue, the
oxygen atoms red and the sulphur atoms
yellow. (C) Same as in A, but rotated )90°
about a vertical axis.
GluR2 ligand-binding core in complex with (S)-4-AHCP B. B. Nielsen et al.
1642 FEBS Journal 272 (2005) 1639–1648 ª 2005 FEBS
space available in the pocket (formed mostly by D1)
forces larger ligands closer to D2.
The seven-membered ring of (S)-4-AHCP fills out
some of the same space as the 2-methyl-tetrazole ring
of (S)-2-Me-Tet-AMPA, but does not protrude as dee-
ply into the pocket. However, unlike the tetrazole ring,
the seven-membered ring is not planar and it occupies
additional space towards the residues Glu402 from
D1 and Thr686 from D2, constituting the lock between
the two domains of the agonist-bound GluR2 ligand-
binding core. Atoms C5, C6 and C7 in particular
approach the lock, but without major disturbance.
This interdomain interaction is still intact in the sense
that a hydrogen bond is formed between the two resi-
dues, however, the distance is somewhat longer (3.1 A
˚
)
than in the other three complexes ( 2.7 A
˚
).
The residues Leu650 and Met708 that are involved
in hydrophobic interactions with the ligands show con-
formational variability in the structures under discus-
sion. The side chain of Leu650 is flipped  180° in

(S)-4-AHCP (v
1
¼ )87°; v
2
¼ )167°) relative to the
(S)-glutamate (v
1
¼ 179°; v
2
¼ 65°; mol C) (S)-2-Me-
Tet-AMPA (v
1
¼ 172°; v
2
¼ 68°; mol B) and (S)-
AMPA (v
1
¼ 179°; v
2
¼ 67°; mol C) complexes and
adopts a conformation more like the one seen in the
GluR2-S1S2J:kainate complex (v
1
¼ )94°; v
2
¼ 166°).
Leu650 apparently adjusts its conformation for opti-
mal hydrophobic contact with (S)-4-AHCP. The side
chain of Met708 lends flexibility to the binding pocket;
its conformation adjusting to fit various ligands

[15,18–20]. In the (S)-4-AHCP complex, the tail of this
side chain skirts the binding pocket to avoid clashing
with the ligand. The Ce atom of Met708 points back
into favourable van der Waals’ contact with the seven-
membered ring of (S)-4-AHCP.
(S)-4-AHCP is a partial agonist at GluR2
The D1–D2 domain closure in the GluR2-S1S2J:(S)-4-
AHCP structure is 16.9° (relative to the structure of apo
GluR2-S1S2J), which is less than the domain closure
for full agonists ( 21°). The apparent explanation for
this is twofold. Firstly (S)-4-AHCP has an extra carbon
atom between the a-amino acid and the distal anionic
moieties compared with other 3-hydroxy isoxazole
analogues. Although conformational restriction shor-
tens the distance between the a-amino acid group and
the distal 3-hydroxy anion, the isoxazole is nonetheless
pushed deeper into D2 than in the other three [(S)-glu-
tamate (S)-AMPA and (S)-2-Me-Tet-AMPA] structures
when adopting the conformation required for recogni-
tion of a-amino acids. This favours intermediate
domain closure. Secondly, driving the domains closer to
each other would result in steric clashes between the
bicyclical ring system of (S)-4-AHCP and the backbone
atoms of Leu704. Also, the side chains of Tyr450,
Thr655, Glu705 and Met708 would need to rearrange.
In the present structure, the Ca atom of Leu704 is
displaced 0.9 A
˚
(Fig. 4B) compared to its position in
GluR2-S1S2J:(S)-glutamate. One consequence of the

position of the isoxazole ring close to D2 is the hydro-
gen bond formed between the isoxazole oxygen atom
and the backbone nitrogen atom of Glu705 (Fig. 3C).
This is different from other known structures of GluR2
in complex with isoxazole-based agonists.
The GluR2-S1S2J:(S)-4-AHCP complex forms a
dimer in the crystal as observed in all other agonist
complexes reported. The dimer is generated by apply-
ing crystallographic symmetry to the monomer that is
observed in the asymmetric unit of the crystal. The dis-
tance between the GT-linker (replacing the M1 and
M2 transmembrane regions) of both protomers has
been shown to be linearly related to the degree
of domain closure [15,18,21]. In this structure, the
Table 2. Residues in GluR2-S1S2J involved in ionic interactions and
hydrogen bonds (< 3.3 A
˚
) with (S )-4-AHCP. Residues and water
molecules within 5 A
˚
from any ligand atom: Glu402,Tyr450,
Pro478, Leu479, Thr480, Arg485, Leu650, Ser652, Gly653, Ser654,
Thr655, Lys656, Thr686, Leu703, Leu704, Glu705, Met708, Tyr732,
W1-W3 and W6-W10.
Conformation 1 (A
˚
) Conformation 2 (A
˚
)
Carboxylate oxygen O3

a
Thr480 N 2.8 2.9
Arg485 Ng1 2.8 2.7
Carboxylate oxygen O4
Ser654 N 2.7 2.9
Ser654 Oc 3.2 (3.5)
Arg485 Ng2 2.8 2.8
Ammonium group N2
Pro478 O 2.9 2.7
Thr480 Oc1 2.9 2.8
Glu705 Oe1 3.1 3.3
Glu705 Oe2 2.6 2.8
Isoxazole oxygen O1
Glu705 N 2.8 3.1
W3
b, c
(3.7) 3.3
Isoxazole nitrogen N1
Thr655 Oc1 2.6 2.8
W2
d
3.0 2.9
3-hydroxy anion O2
Thr655 N 3.3 3.3
W1
e
2.6 2.9
a
For atom numbering, see Fig. 1.
b

Numbering of water molecules
as per Kasper et al. [19].
c
W3 is further hydrogen bonded to the
side chains of Thr686 and Tyr702.
d
W2 also interacts with the
backbone of residues Leu650 and Leu703.
e
W1 is further connec-
ted to the backbone of residues Ser652, Thr655 and Lys656.
B. B. Nielsen et al. GluR2 ligand-binding core in complex with (S)-4-AHCP
FEBS Journal 272 (2005) 1639–1648 ª 2005 FEBS 1643
distance is 34.1 A
˚
(between Ile633-Ile633 Ca-atoms). It
has been suggested previously that the movement of
D2 to close over the ligand causes conformational
strain that is transferred to the ion channel, leading to
pore opening [27]. (S)-4-AHCP has an efficacy of 0.38
at GluR2(Q)
i
relative to (S)-glutamate. The efficacy
combined with the observed domain closure (16.9°)
and D2–D2 linker separation imply that (S)-4-AHCP
acts as a partial agonist at the AMPA-type receptor
GluR2. Based on comparisons with structural and
pharmacological studies on a range of other agonists
[15,18,21], partial agonism was indeed expected from
the observed degree of GluR2-S1S2J domain closure

and D2–D2 linker separation.
(S)-4-AHCP and receptor subtype selectivity
(S)-4-AHCP displays selectivity for homomers of the
low affinity kainate receptor subunit GluR5 over the
AMPA receptors [26]. The most important differences
in the ligand-binding site between GluR2 and GluR5
are the respective substitutions of Leu650 and Met708
in GluR2 to the smaller residues Val685 and Ser741 in
GluR5 (numbering as in TrEMBL entry Q86SU9). In
particular, mutagenesis studies have shown that the
latter residue is responsible for the selectivity displayed
by another GluR5 subtype selective agonist (S)-
2-amino-(5-tert-butyl-3-hydroxy-4-isoxa zolyl)propionic
acid [28]. Recently, Armstrong et al. [29] reported that
the mutation of Leu650 to Thr in GluR2 yields a
receptor that responds more potently and efficaciously
to the partial agonist kainate and less to the full agon-
ist AMPA compared to unmodified GluR2. Also, the
nonconserved residue at position 702 in GluR2 has
been identified as the major contributor to the selectiv-
ity of (S)-Br-HIBO for GluR1 (Tyr698) over GluR3
(Phe706) [30]. In GluR5, this residue is Leu735 and it
may thus play a role in receptor selectivity. Thr686
A
B
Fig. 4. Comparison of the GluR2-S1S2J:
(S)-4-AHCP complex with other agonist
complexes. (A) Superposition of the struc-
tures of GluR2-S1S2J in complex with:
(S)-4-AHCP (grey) (S)-glutamate (yellow; pdb

code 1FTJ, mol C) (S)-AMPA (cyan; pdb
code 1FTM, mol C) and (S)-2-Me-Tet-AMPA
(magenta; pdb code 1M5B, mol B), shown
in stereo. Selected residues of the ligand-
binding site are shown. Superimposition of
the Ca atoms of D1 (residues 393–496 and
730–773) of the three structures on the
(S)-4-AHCP complex resulted in rmsd of
0.36, 0.33 and 0.80 A
˚
, respectively. The
spheres represent water molecules,
the nitrogen atoms are coloured blue, the
oxygen atoms red and the sulfur atoms
yellow. (B) Ca-trace of the structures of
GluR2-S1S2J:(S)-4-AHCP (grey) and of
GluR2-S1S2 J:(S)-glutamate (yellow), super-
imposed by Ca atoms of D1. (S )-4-AHCP
(conformation 1) and the side chain of
Leu704 are shown in ball-and-stick.
GluR2 ligand-binding core in complex with (S)-4-AHCP B. B. Nielsen et al.
1644 FEBS Journal 272 (2005) 1639–1648 ª 2005 FEBS
forms an interdomain interaction with Glu402, and is
in close contact with atoms of the seven-membered
ring system; this residue is replaced by the smaller
Ser721 in GluR5.
Homology modelling of the ligand-binding core of
GluR5 and docking of (S)-4-AHCP to this model has
shown that the binding site is larger than in GluR2;
thus allowing the accommodation of larger and more

bulky ligands [26]. A corresponding analysis based on
the structure of GluR2-S1S2J in complex with (S)-4-
AHCP supports this finding. Using single-channel
recordings, domain closure has elegantly been shown
to correlate with the open probability of discrete
subconductance states of the channel and also with
receptor desensitization [21]. Taken together, the
smaller residues in GluR5 would probably allow for
increased domain closure compared to that of GluR2,
as well as a more complementary van der Waals’
environment, and this may explain the functional
selectivity of (S)-4-AHCP towards GluR5. A fuller
understanding of GluR5 selectivity awaits the publica-
tion of the structure of a GluR5 construct in complex
with an agonist.
Experimental procedures
Materials
Chemicals were purchased from Sigma-Aldrich (Vallensbæk
Strand, Denmark) unless otherwise specified. The synthesis
of (S)-4-AHCP is as described by Brehm et al. [26]. Restric-
tion and other molecular biological enzymes were obtained
from New England BioLabs (Beverley, MA, USA).
Protein expression and purification
The GluR2-S1S2J construct described by Armstrong and
Gouaux [15] was expressed, refolded and purified essentially
as previously reported [17,31].
The rat AMPA receptor clone GluR2(Q)
i
within the vector
pGEMHE [32] was used for preparation of high-expression

cRNA transcripts. cDNA were grown in XL1 Blue bacteria
(Stratagene, La Jolla, CA, USA) and prepared using column
purification (Qiagen, Hilden, Germany). cRNA was synthes-
ized from this cDNA using the mMessage mMachine T7
mRNA-capping transcription kit (Ambion Inc., Austin, TX,
USA).
Cell culture
Sf9 insect cells were maintained in BaculoGold Max-XP
serum-free medium (BD Biosciences, FranklinLakes, NJ,
USA) according to standard manufacturers protocols.
Receptor binding assay
(S)-4-AHCP binding affinity at the GluR2-S1S2J soluble
construct and at full-length GluR2(R)
o
was determined by a
radioligand binding assay. Purified construct (0.1 lg protein)
or Sf9 insect cell membranes (0.2–0.4 mg protein) expressing
GluR2(R)
o
[33] were incubated with 2–4 nm (RS)-
[5-methyl-
3
H]-AMPA (43.5 CiÆmmol
)1
; Perkin Elmer, Well-
esley, MA, USA) in the presence of 1 nm)0.10 mm (S)-4-
AHCP for 1–2 h on ice in assay buffer (50 mm Tris ⁄ HCl,
100 mm KSCN, 2.5 mm CaCl
2
, pH 7.2 at 4 °C; containing

10% glycerol for GluR2-S1S2J). Samples were filtered onto
Millipore 0.22-lm GSWP nitrocellulose filters (for GluR2-
S1S2J) or Whatman GF ⁄ B filters [for GluR2(R)
o
]. Filters
were washed twice with cold assay buffer and radioactivity
was determined by scintillation counting. Data were analysed
using grafit v3.00 (Erithacus Software Ltd, Horley, UK)
and fit as previously described [34] to determine Hill coeffi-
cient and K
i
. The K
d
values of [
3
H]AMPA at GluR2-S1S2J
(12.8 nm) and GluR2(R)
o
(16.8 nm) were determined previ-
ously [18,33].
Electrophysiology
All frog experimental procedures are approved by the
Experimental Animal Committee, The Danish Ministry of
Justice, Copenhagen, Denmark (2004/561-876-C10). Mature
female X. laevis (African Reptile Park, Tokai, South Africa)
were anesthetized using 0.1% ethyl 3-aminobenzoate, meth-
anesulfonic acid salt (tricaine methanesulfonic acid salt)
by transdermal administration and ovaries were surgically
removed. The ovarian tissue was dissected and treated with
1mgÆmL

)1
collagenase in nominally Ca
2+
-free Barth’s
medium for 2 h at room temperature. On the second day,
oocytes were injected with 50 nL ( 1 lgÆlL
)1
) cRNA and
incubated in Barth’s medium (88 mm NaCl, 1 mm KCl,
0.33 mm Ca(NO
3
)
2
, 0.41 mm CaCl
2
, 0.82 mm MgSO
4
,
2.4 mm NaHCO
3
,10mm Hepes, pH 7.4) with 0.1 mgÆmL
)1
gentamicin and 1% penicillin–streptomycin (Life Technol-
ogies) at 17 °C. Oocytes were typically used for recordings
from 3 to 10 days postinjection and were voltage-clamped
with the use of a two-electrode voltage clamp (GeneClamp
500B, Axon Instruments, Union City, CA, USA) with both
microelectrodes filled with 3 m KCl. Recordings were made
while the oocytes were continuously superfused with nomin-
ally Ca

2+
-free frog Ringer’s solution (115 mm NaCl, 2 mm
KCl, 1.8 mm BaCl
2
,5mm Hepes, pH 7.0). Drugs were dis-
solved in Ca
2+
-free frog Ringer’s solution and added by
bath application. Recordings were made at room tempera-
ture at holding potentials in the range of )80 to )20 mV.
For efficacy measurements, (S)-4-AHCP was applied at a
saturating concentration (500 lm) in the presence of
100 lm cyclothiazide in order to block receptor desensitiza-
tion (cyclothiazide EC
50
: GluR2(Q)
i
¼ 7.6 lm [35]). Control
B. B. Nielsen et al. GluR2 ligand-binding core in complex with (S)-4-AHCP
FEBS Journal 272 (2005) 1639–1648 ª 2005 FEBS 1645
stimulations with 1 mm (S)-glutamate plus 100 lm cyclothi-
azide were performed immediately prior to, and after (S)-4-
AHCP application, with a washout period of 5–10 min
between drug applications. The two control (S)-glutamate
stimulations were each no more than 1–9% different from
the mean value. Cyclothiazide (100 lm) was preapplied
alone for 1 min before each agonist application. The (S)-4-
AHCP maximum response was then expressed as a fraction
of the mean value of the two test (S)-glutamate stimula-
tions.

Data analysis of pharmacology
Student’s t-test was used for comparison of K
i
values using
sigmastat for Windows v3.0 (SPSS Inc., Chicago, IL,
USA). Values are given as mean ± SEM and were consid-
ered statistically significantly different if P < 0.05. Concen-
tration–response curves for agonists were analysed using
grafit v3.00 to determine the EC
50
and Hill value (n
H
),
using Eqn (1), where I is the measured current and I
max
is
the maximal steady-state current.
I ¼ I
max
=ð1 þ 10
ðlog½EC
50
Þ=10
ðlog½AgonistÞ
Þ
n
H
ð1Þ
Co-crystallization of GluR2-S1S2J with
(S)-4-AHCP

The GluR2-S1S2J protein was dialysed extensively in the
buffer used for crystallization (10 mm Hepes pH 7.0, 20 mm
NaCl, 1 mm EDTA) and concentrated to 6 mgÆmL
)1
. The
GluR2-S1S2J was mixed with (S)-4-AHCP at a ratio of
1 : 49. Crystals were obtained at 6 °C by the hanging drop
vapour diffusion method using a reservoir solution contain-
ing 0.2 m lithium sulfate, 0.1 m phosphate–citrate buffer
pH 4.5 and 20% PEG 3350. Crystals were transferred
through a cryo-protectant solution consisting of 4.7 mm
ligand and 12% glycerol in reservoir solution prior to
flash-cooling.
X-ray data collection
The X-ray diffraction data were collected from one crystal
at 100 K and at a wavelength of 0.811 A
˚
using a MAR
CCD detector at beamline X11 (DESY, Hamburg, Ger-
many). The crystal diffracted to 1.75 A
˚
. The HKL package
(Denzo and Scalepack) [36] was used for autoindexing and
data processing, for statistics see Table 1.
Structure determination and refinement
The structure was solved using molecular replacement with
amore [37] implemented in the ccp4i package [38]. The
protein atoms of the structure of GluR2-S1S2J complexed
with (S)-Br-HIBO ([18]; pdb code 1M5C), was used as a
search model. Only one solution to both the rotation- and

translation function was obtained. The program arp ⁄ warp
[39] was used for tracing the majority of the structure.
Refinements alternating with manual model building were
performed using the programs refmac5 [40] and o [41],
respectively.
The electron density corresponding to (S)-4-AHCP was
well defined and allowed unambiguous positioning of two
different conformations of the ligand (refined with equal
occupancy; see Fig. 3A). Initially (S)-4-AHCP was mod-
elled as a single conformation; however, additional F
o
–F
c
difference electron density was present, indicating conform-
ationally enantiomeric puckering of the seven-membered
ring of the ligand. The atoms of the seven-membered ring
were built and refined in two conformations but additional
density still appeared. Therefore, two conformations (inclu-
ding all ligand atoms) were modelled and all atoms refined
with half occupancy. This resulted in the disappearance of
the additional difference electron density.
A monomer library description of the ligand for REF-
MAC5 has been created. (S)-4-AHCP was built as the tri-
ion and submitted to conformational analysis using the
MMFFs force field with GB-SA treatment of solvation in
macromodel 8.1 [42]. Three distinct low energy conform-
ers were chosen to represent the repertoire of the ring and
truncated systems were built (conformers of 4-ethyl-7,8-
dihydro-6H-cyclohepta[d]isoxazol-3-ol anion). These were
minimized using Density Functional Theory [B3LYP ⁄ 6–

311 + G(d,p)] in gaussian¢03 [43] to give highly accurate
co-ordinates for the ring system. The amino-acid group was
rebuilt from the ethyl side chain and the resulting glycine
moiety was re-minimized with MMFFs ⁄ GB-SA with the
other atoms frozen in the positions determined by quantum
chemistry. The coordinates of one of the low energy con-
formations of (S)-4-AHCP were used for the library des-
cription. Water molecules as well as two sulfate ions and
three glycerol molecules were included as refinement
progressed.
The refined structure comprises (using the numbering of
full-length membrane bound receptor without signal pep-
tide, Swiss-Prot entry P19491) residues 392–506, the GT
linker and residues 632–774, as well as two additional
N-terminal residues. For refinement statistics (Table 1). The
coordinates of the GluR2-S1S2J structure in complex with
(S)-4-AHCP have been deposited in the RCSB Protein
Data Bank with accession code 1WVJ.
Structure analysis and figure preparation
The hingefind script [44] implemented in the program
vmd [45] was used to calculate the ligand-induced domain
closure relative to the apoGluR2-S1S2J structure (pdb code
1FTO, mol A). The CCP4 program contacts was used in
the analysis of protein–ligand interactions. The programs
molscript [46], raster3d [47] and bobscript [48] were
used in the preparation of figures.
GluR2 ligand-binding core in complex with (S)-4-AHCP B. B. Nielsen et al.
1646 FEBS Journal 272 (2005) 1639–1648 ª 2005 FEBS
Acknowledgements
L. B. Sørensen is kindly acknowledged for technical

assistance. This work was supported by: DANSYNC
(Danish Centre for Synchrotron Based Research); the
Danish Medical Research Council; the Novo Nordisk
Foundation; the Lundbeck Foundation; the computing
resources of the Australian Centre for Advanced Com-
puting and Communications as well as the Danish
Center for Scientific Computing; and the European
Community – Access to Research Infrastructure
Action of the Improving Human Potential Programme
to the EMBL Hamburg Outstation, contract number
HPRI-CT-1999-00017.
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