Exploring the GluR2 ligand-binding core in complex with the bicyclical AMPA analogue (S)-4-AHCP

Exploring the GluR2 ligand-binding core in complexwith the bicyclical AMPA analogue (S)-4-AHCPBettina B. Nielsen1, Darryl S. Pickering2, Jeremy R. Greenwood1, Lotte Brehm1, Michael Gajhede1,Arne Schousboe2 and Jette S. Kastrup1

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)propionic

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: [email protected]

(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 Ki

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; EC50, 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; nH, 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): Ki ¼ 185 ± 29 nm; Hill

coefficient (nH) ¼ 1.03 ± 0.04 (n ¼ 4) and for full-

length GluR2(R)o: Ki ¼ 175 ± 8 nm; nH ¼ 0.95 ± 0.02

(n ¼ 3) (Fig. 2A). No statistically significant difference

between the Ki 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 (EC50) ¼17.5 ± 1.2 lm, nH 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 EC50 values have been reported for

(S)-4-AHCP at the other AMPA receptors: GluR1o(4.5 lm), GluR3o (7.2 lm) and GluR4o (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 [3H]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 lM CTZ; white box) was preapplied for 60 s

before application of 1 mM (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 P21212

Unit cell parameters (A) a ¼ 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)

Rsym (%) 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

Rwork (%) 16.9 (24.8)

Rfree, 5% (%) 21.4 (31.8)

Rms deviations

Bond lengths (A) 0.017

Bond angles (�) 1.6

Residues in allowed regions

of Ramachandran plot (%)b99.6

Mean B-values (A2)

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


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-2H-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) 2Fo-Fc 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 Fo-Fc

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.



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 (v1 ¼ )87�; v2 ¼ )167�) relative to the

(S)-glutamate (v1 ¼ 179�; v2 ¼ 65�; mol C) (S)-2-Me-

Tet-AMPA (v1 ¼ 172�; v2 ¼ 68�; mol B) and (S)-

AMPA (v1 ¼ 179�; v2 ¼ 67�; mol C) complexes and

adopts a conformation more like the one seen in the

GluR2-S1S2J:kainate complex (v1 ¼ )94�; v2 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 O3a

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

W3b, c (3.7) 3.3

Isoxazole nitrogen N1

Thr655 Oc1 2.6 2.8

W2d 3.0 2.9

3-hydroxy anion O2

Thr655 N 3.3 3.3

W1e 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.



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-isoxazolyl)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

BFig. 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.



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-3H]-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 CaCl2, pH 7.2 at 4 �C; containing10% 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 Ki. The Kd values of [3H]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

1 mgÆmL)1 collagenase in nominally Ca2+-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(NO3)2, 0.41 mm CaCl2, 0.82 mm MgSO4,

2.4 mm NaHCO3, 10 mm 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 Ca2+-free frog Ringer’s solution (115 mm NaCl, 2 mm

KCl, 1.8 mm BaCl2, 5 mm Hepes, pH 7.0). Drugs were dis-

solved in Ca2+-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 EC50: GluR2(Q)i ¼ 7.6 lm [35]). Control



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 Ki 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 EC50 and Hill value (nH),

using Eqn (1), where I is the measured current and Imax is

the maximal steady-state current.

I ¼ Imax=ð1þ 10ðlog½EC50 �Þ=10ðlog½Agonist�ÞÞnH 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 Fo–Fc

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.



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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Documents

Exploring the GluR2 ligand-binding core in complex with the bicyclical AMPA analogue (S)-4-AHCP