Lessons from crystal structures of kainate receptors

Accepted Manuscript Lessons from crystal structures of kainate receptors Stine Møllerud, Karla Frydenvang, Darryl S. Pickering, Jette Sandholm Kastrup PII:

S0028-3908(16)30212-X

DOI:

10.1016/j.neuropharm.2016.05.014

Reference:

NP 6315

To appear in:

Neuropharmacology

Received Date: 29 April 2016 Revised Date:

19 May 2016

Accepted Date: 22 May 2016

Please cite this article as: Møllerud, S., Frydenvang, K., Pickering, D.S., Kastrup, J.S., Lessons from crystal structures of kainate receptors, Neuropharmacology (2016), doi: 10.1016/ j.neuropharm.2016.05.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Neuropharmacology – Review

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Lessons from crystal structures of kainate receptors

Stine Møllerud, Karla Frydenvang, Darryl S. Pickering and Jette Sandholm Kastrup*

Department of Drug Design and Pharmacology, Faculty of Health and Medical

* Contact Information:

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Sciences, University of Copenhagen, Jagtvej 162, DK-2100 Copenhagen Ø, Denmark

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Email: [email protected], phone: +45 3533 6486

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ACCEPTED MANUSCRIPT Highlights

Kainate receptors belong to the family of ionotropic glutamate receptors

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These receptors are important for memory and learning

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A review on 84 crystal structures of kainate receptors is presented

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We discuss binding of agonists, antagonists, ions and mutations

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Abstract

Kainate receptors belong to the family of ionotropic glutamate receptors. These

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receptors assemble from five subunits (GluK1-5) into tetrameric ion channels. Kainate receptors are located at both pre- and postsynaptic membranes in the central nervous system where they contribute to excitatory synaptic transmission and modulate network excitability by regulating neurotransmitter release. Dysfunction of

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kainate receptors has been implicated in several neurological disorders such as epilepsy, schizophrenia and depression. Here we provide a review on the current understanding of kainate receptor structure and how they bind agonists, antagonists

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and ions. The first structure of the ligand-binding domain of the GluK1 subunit was reported in 2005, seven years after publication of the crystal structure of a soluble

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construct of the ligand-binding domain of the AMPA-type subunit GluA2. Today, a full-length structure has been determined of GluK2 by cryo electron microscopy to 7.6 Å resolution as well as 84 high-resolution crystal structures of N-terminal domains and ligand-binding domains, including agonist and antagonist bound structures, modulatory ions and mutations. However, there are still many unanswered questions and challenges in front of us.

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ACCEPTED MANUSCRIPT Graphical abstract

Kainate receptors contribute to fast excitatory neurotransmission and have been linked to brain diseases. We provide a review on 84 crystal structures of N-terminal and

Chemical compounds studied in this article

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ligand-binding domains of kainate receptors.

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ATPO (PubChem CID 4615193), domoic acid (PubChem CID 5282253),

dysiherbaine (PubChem CID 9839436), L-glutamic acid (PubChem CID 33032),

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kainic acid (PubChem CID 10255), LY466195 (PubChem CID 10168249 ), (2S,4R)4-methylglutamic acid (PubChem CID 95883), neodysiherbaine A (PubChem CID 11460505), quisqualic acid (PubChem CID 1209), UBP310 (PubChem CID 6420160)

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Keywords

Kainate receptors, N-terminal domain, ligand-binding domain, crystal structures, ligands, mutations

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Abbreviations

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AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; iGluRs, ionotropic glutamate receptors; LBDs, ligand-binding domains; NMDA, N-methyl-D-aspartate; NTDs, N-terminal domains

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ACCEPTED MANUSCRIPT 1. Introduction

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and exerts its fast effects through ionotropic glutamate receptors (iGluRs) by

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gating of their cationic channels to generate synaptic current essential to brain function. The iGluRs are grouped into four classes based on their sequence similarity and preferred agonist: N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-

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4-isoxazolepropionate (AMPA), kainate and delta receptors (Traynelis et al., 2010). It is a characteristic feature of these receptors that they form tetrameric ion channels.

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The kainate receptors assemble from five subunits (GluK1-5) whereof GluK1-3 (previously named GluR5-7) can form homomeric and heteromeric receptors (Egebjerg et al., 1991, Schiffer et al., 1997). In contrast, GluK4-5 (previously known as KA-1 and KA-2, respectively) require heteromeric assembly with one of the

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GluK1-3 subunits to form functional channels (Herb et al., 1992, Werner et al., 1991). GluK1-3 have been termed the low-affinity kainate receptors and GluK4-5 the highaffinity kainate receptors (Fletcher and Lodge, 1996). Unlike AMPA receptors that

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are found exclusively at postsynaptic sites, kainate receptors are located at both pre-

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and postsynaptic membranes. Here, they contribute to excitatory synaptic transmission and modulate network excitability by regulating neurotransmitter release (Contractor et al., 2011). Owing to this involvement in neuronal function, dysfunction of kainate receptors have been implicated in several neurological disorders such as epilepsy, schizophrenia, depression and bipolar disorder (Das et al., 2012; Ibrahim et al., 2000; Li et al., 2010; Milanesi et al., 2015; Pickard et al., 2006).

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ACCEPTED MANUSCRIPT We here present a review of 84 crystal structures of kainate receptor N-terminal domains and ligand-binding domains deposited in the Protein Data Bank (www.pdb.org) as of April 2016. The structures have provided functional insight at

antagonists as well as cations and anions.

2. GluK2 full-length structure

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the molecular level and revealed the binding modes of several agonists and

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The extracellular part of the receptor is comprised of N-terminal domains (NTDs) that exist as dimers-of-dimers with two-fold symmetry and are proximal to the ligand-

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binding domains (LBDs) that harbor the binding site for (S)-glutamate (Fig. 1A). The LBDs also form dimers-of-dimers but a crossover occurs from the NTD layer to the LBD layer, meaning that different subunits form the NTD and LBD dimers. The ion

loops.

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channel pore located in the membrane is composed of 12 α-helices and four P-entrant

Whereas full-length crystal structures are available of the AMPA receptor GluA2

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(Sobolevsky et al., 2009; Chen et al., 2014; Dürr et al., 2014, Yelshanskaya et al., 2014) and the NMDA receptor GluN1/GluN2B (Karakas and Furukawa, 2014; Lee et

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al., 2014), no full-length crystal structures have been reported of kainate receptors so far. However, an EM structure of a tetrameric full-length GluK2 in complex with the agonist (2S,4R)-4-methylglutamate has been published where the resolution extends to 7.6 Å (PDB code 4UQQ; Meyerson et al., 2014). Furthermore, single-particle cryoelectron tomography structures were reported by Schauder et al. (2013) showing subunit crossover in the resting state.

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ACCEPTED MANUSCRIPT The full-length GluK2 structure was suggested to resemble the desensitized state, which is a state where the channel is closed even though glutamate/agonist is still bound to the receptor (Fig. 1A). In this desensitized GluK2 the NTD dimer structure is conserved (Fig. 1B) compared to a soluble NTD dimer, whereas drastic changes

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have occurred in the LBD dimer with a ~125° rotation of the LBDs with respect to

closure.

3. Structures of N-terminal domains

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each other (Fig. 1C). This conformational change was suggested to lead to channel

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The N-terminal domain is primarily of importance for assembly of the tetrameric receptor and is thought to serve a modulatory role. Today, structures have been reported of the NTD of GluK2, GluK3 and GluK5, whereas, surprisingly, structures have not been reported of the otherwise well-characterized GluK1 and also not of

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GluK4 (Table 1).

Kumar et al. (2009) demonstrated that the GluK2-NTD forms dimers in solution at

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micromolar protein concentrations and also crystallizes as a dimer (Fig. 2). Each NTD subunit was seen to adopt an intermediate degree of lobe R1-R2 closure compared to

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the apo and ligand-bound complexes of Leucine/Isoleucine/Valine Binding Protein and G protein-coupled glutamate receptors. This observation was further substantiated by the structure determinations of the GluK3-NTD and GluK5-NTD (Kumar and Mayer, 2010), showing an up to 10° variation among kainate receptors compared to 50° at the NMDA receptor subunit GluN2B. The limited lobe movement in kainate receptor NTDs was suggested to result from extensive interlobe contacts between R1 and R2 and dimers assembly, where both the R1 and R2 lobes form extensive contacts.

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ACCEPTED MANUSCRIPT Compared to the GluK2-NTD dimer, the GluK3-NTD dimer is almost identical while GluK5-NTD forms a different dimer (Kumar and Mayer, 2010). Interestingly, Kumar et al. (2011) showed that the GluK2-NTD co-assembles with the GluK5-NTD with a Kd of 11 nM; a 32,000-fold better Kd than for formation of the GluK5-NTD

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homodimer, high-lighting the importance of the NTD for the formation of heteromeric kainate receptors.

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4. Ligand-binding domain structures

To date, LBD structures have been reported of all three low-affinity kainate receptors,

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GluK1-3, whereas no structures have so far been published of the high-affinity kainate receptor subunits GluK4 and GluK5. However, we have recently been successful in determining the structure of the GluK4-LBD (to be published). The LBD is comprised of a clamshell structure, composed of two lobes D1 and D2 (Fig.

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3A). The orthosteric ligand (S)-glutamate, as well as agonists and competitive antagonists, binds at a site located between the two lobes. An overlay of the LBD

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4.1. Agonists

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structures of GluK1-3 shows that the overall architecture is very similar (Fig. 3B).

Until now, structures of GluK1-LBD have been determined in complex with ten different agonists, GluK2-LBD with seven agonists and GluK3-LBD with six agonists. These structures have provided insight into the molecular mechanism of binding and indirect information on receptor activation. The structure determinations have also been part of drug discovery programs in the search for selective agonists. Whereas selective agonists are valuable pharmacological tool compounds, they will probably not become drugs due to excitotoxicity issues.

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ACCEPTED MANUSCRIPT Glutamate. In 2005, the first structures of kainate receptors were published, showing the detailed binding mode of (S)-glutamate in GluK1-LBD (Mayer, 2005; Naur et al., 2005) and GluK2-LBD (Mayer, 2005). Today, all three low-affinity kainate receptors, GluK1-3, have been crystallized with (S)-glutamate. Five structures of GluK1-LBD

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with glutamate have been determined, three of rat origin and two of human (Table 2). Similarly, more than one structure has been determined of GluK2-LBD and GluK3-

LBD in complex with glutamate, i.e. four rat GluK2-LBD and three rat GluK3-LBD

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structures (Table 2). The binding mode of glutamate is similar in all structures.

Therefore, only the binding of glutamate in GluK1-LBD will be described in detail,

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and differences among the subunits discussed.

Upon binding, glutamate adopts an anti and gauche(-) conformation around the central Cα-Cβ and Cβ-Cγ bonds, respectively. A similar conformation of glutamate is

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seen when binding to AMPA receptors (Armstrong and Gouaux, 2000). The αcarboxylate group of glutamate interacts with the essential D1 residue Arg523 side chain (GluK1-2 numbering including signal peptide) via salt bridges and further

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makes charge-assisted hydrogen bonds to the main chain nitrogen atoms of Thr518 in D1 and Ser689 in D2 (Fig. 4A). The α-ammonium group forms a salt bridge with the

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side chain of Glu738 in D2 as well as three charge-assisted hydrogen bonds to the backbone oxygen of Pro516 in D1, the side chain hydroxyl group of Thr518 in D1 and water molecule W1. The distal γ-carboxylate is engaged in a hydrogen bonding network potentially involving both D2 residues and water molecules: charge-assisted hydrogen bonds to backbone nitrogen atoms of Ser689 and Thr690, side chain hydroxyl group of Thr690 and three water molecules (W2-4).

Within 4 Å of glutamate, all residues are the same in rat and human GluK1 and the

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ACCEPTED MANUSCRIPT binding mode of glutamate is very similar. Also, only few differences in binding site residues are observed in GluK1-3. A total of ten residues (D1: Tyr489, Pro516, Leu517, Thr518 and Arg523; D2: Gly688, Ser689, Thr690, Glu738 and Tyr764) are located within 4 Å of glutamate in GluK1-LBD, of which only Thr518 and Ser689

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differ among the three receptors (Fig. 4B). Whereas Thr518 is also a threonine in

GluK3, it corresponds to an alanine in GluK2. Further, in GluK2-3 alanine is seen at

the position of Ser689 in GluK1. Considering the nearest surroundings of glutamate,

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this creates a slightly larger binding site in GluK2 compared to GluK1 and at the same

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time eliminates polar contacts to the side chain of Ala518 in GluK2.

One additional important difference that should be mentioned is Ser721 in D2 of GluK1 that is involved in the formation of a D1-D2 interlobe contact to Glu441 in D1 in structures with agonists (Fig. 4B). This residue corresponds to an asparagine in

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GluK2-3. The presence of this larger residue in GluK2-3 exposes many ligands to steric hindrance, probably explaining why it has been easiest to obtain GluK1 preferring agonists and antagonists.

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Kainate and domoate. Kainate receptors were first identified as a distinct iGluR

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receptor class when it was discovered in late 70’s - beginning of the 80’s that they could be activated by low concentrations of kainate, a natural compound isolated from the red alga Digenea simplex in 1953. In 2005, the first structure of a kainate receptor in complex with kainate was reported, i.e. GluK2-LBD (Mayer, 2005). Later followed structures of kainate with GluK1-LBD and GluK3-LBD (Table 2). Kainate is seen to form similar contacts as glutamate to binding site residues (Fig. 5A); however, the pyrrolidine ring of kainate displaces W1. The 4-isopropenyl group of kainate is directed towards Glu441 and Ser721 in GluK1 and thereby weakens the D1-D2

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ACCEPTED MANUSCRIPT interlobe contact (Fig. 5A). This is not the case in the structures of GluK2-LBD and GluK3-LBD with kainate, where a hydrogen bond is still seen between Glu440 and Asn721 (GluK2 numbering). Thus, this might explain the slightly lower binding

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affinity of kainate at GluK1-LBD compared to GluK2-3 (Table 2). Domoate is an α-amino acid metabolite produced by certain species of phytoplankton and algae, with a chemical structure resembling that of kainate (Table 2). The

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structure of GluK2-LBD in complex with domoate was determined in 2005 (Nanao et al., 2005), and later followed the structure of domoate in GluK1-LBD (Hald et al.,

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2007). The hexa-1,3-dienyl moiety of domoate extends out of the binding site, where the carboxylate group forms a hydrogen bond to the backbone nitrogen atom of Tyr489 in GluK1 (Fig. 5B). Otherwise, contacts are similar to those of kainate.

Dysiherbaines. Another class of agonists that has been extensively studied is the

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marine natural product dysiherbaine and analogues isolated from natural sources or prepared by total synthesis (Table 2). In total, six different compounds were crystallized with GluK1-LBD (dysiherbaine (Fig. 5C), 8-deoxy-neodysiherbaine A, 9-

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deoxy-neodysiherbaine A, 8-epi-neodysiherbaine A, neodysiherbaine A and MSVIII-

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19), and one of these (neodysiherbaine A) also in GluK2-LBD. These compounds range from very weak partial agonists/functional antagonists (9-deoxyneodysiherbaine A, MSVIII-19) to highly efficacious partial agonists or full agonists (dysiherbaine, neodysiherbaine A and 8-deoxy-neodysiherbaine A) (Lash et al., 2008; Sanders et al., 2005). However, not all synthetic analogues of dysiherbaine or neodysiherbaine A are described as agonists. For example, 2,4-epi-neodysiherbaine A was defined as an antagonist (or possibly a very weak partial agonist similar to MSVIII-19).

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ACCEPTED MANUSCRIPT Unno et al. (2011) found that differences in three amino acids (Thr518, Ser721 and Ser741 in GluK1 and Ala518, Asn721 and Thr741 in GluK2) led to differences in the binding modes of neodysiherbaine A at GluK1-LBD and GluK2-LBD. Furthermore, deletion of the C9 hydroxyl group in neodysiherbaine A altered the conformation of

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the ligand, making binding less suitable in the GluK1 binding site. It was suggested

that selectivity differences of the dysiherbaine analogues were a result of differences

in the binding mode of the ligands in GluK1 and GluK2 as well as steric repulsion of

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Asn721 in GluK2. Interestingly, despite a similar binding mode of dysiherbaine and

MSVIII-19 in GluK1-LBD, MSVIII-19 acts as a functional antagonist (Frydenvang et

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al., 2009). It was proposed that a weaker stability of the complex compared to that of dysiherbaine might account for the extremely weak agonist efficacy but potent functional antagonist activity of MSVIII-19.

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Other agonists. Finally, the LBDs of GluK1-3 have been crystallized with various other agonists in order to address other scaffolds, conformationally restricted analogues and ligand selectivity (Table 2). For example, the GluK2-LBD was

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crystallized with the potent agonist (2S,4R)-4-methylglutamate that has been extensively used in radiolabel binding affinity studies and quisqualate that occurs

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naturally in the seeds of Quisqualis species.

Binding site water molecules. Seven binding site water molecules (W1-7) are located within 5 Å of glutamate in GluK1-LBD (Fig. 6), of which W1-W6 are also seen in GluK2-LBD and GluK3-LBD. In one of the GluK3 structures, W7 has also been modeled into the binding site (Venskutonyte et al., 2011a). W1 forms direct contact to the α-ammonium group of glutamate and W2-4 contacts to the distal carboxylate. W5-7 do not form direct contacts to glutamate. W5 is engaged in

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ACCEPTED MANUSCRIPT hydrogen bonding to Glu738 and Ser741 as well as to W1 and W2, whereas W6 forms contacts to Ser689, Thr690, Glu738 and a water molecule further away of glutamate. The water molecule W7 makes a hydrogen bond with Ser721 that corresponds to an asparagine in GluK2 and GluK3 as well as with W2 and W3.

W6 are present in crystal structures with agonists.

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4.2. Antagonists

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Agonists can displace water molecules W1, W2, W5 and W7, whereas W3, W4 and

Remarkably, GluK1-LBD only has been crystallized in complex with antagonists,

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hinting that it might be difficult to form crystals of GluK2-LBD and GluK3-LBD with antagonists. In addition, the dearth of antagonists that bind with high affinity to these subunits might also be a contributing factor. A total of 12 different antagonists have been crystallized with GluK1-LBD, of which five belong to the UBP class of

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antagonists (28-32, Table 3). The structures reveal ligand specific conformational changes that complicate rational drug design and underline the importance of

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obtaining experimental structures.

As observed for agonists, the antagonist (S)-1-(2'-amino-2'-carboxyethyl)-3-[(2-

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carboxythien-3-yl)methyl]thieno[3,4-d]pyrimidin-2,4-dione (Table 3) is seen to form a salt bridge between the ligand’s nitrogen atom and the side chain carboxylate of Glu738 (Fig. 7A) as do the antagonists (S)-2-amino-3-(2-(2-carboxyethyl)-5-chloro-4nitrophenyl)propionic acid, (S)-ATPO and CNG10111. It is a characteristic of the UBP compounds that they do not form tight interaction from the ligands nitrogen atoms to the side chain carboxylate of Glu738 (Fig. 7B). Whereas UBP315 has a contact of 3.3 Å, the remaining UBP compounds show distances above 4 Å. This difference might be explained by movement of lobe D2 away from the ligand,

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ACCEPTED MANUSCRIPT resulting in differences in the conformation of the Glu738 side chain (Fig. 7B). The antagonists LY466195 and (3S,4aS,6S,8aR)-6-[3-chloro-2-(1H-tetrazol- 5yl)phenoxy]deca-hydroisoquinoline-3-carboxylic acid also do not form an optimal contact to Glu738 but the most likely explanation here is that the conformation of the

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bicyclic ring system prevents this contact (Fig. 7C). Lastly, binding of the antagonist (S)-2-amino-4-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-6-yl)butanoic acid leads to

extensive variation in lobe D2 movements. All antagonists interfere with the D1-D2

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interlobe contact in GluK1, dramatically increasing the distance between Glu441 in

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D1 and Ser721 in D2 (Fig. 7A).

4.3 Lobe D1-D2 domain closure and interaction

The current model of kainate receptor activation by glutamate or other agonists is that this is driven by a conformational change in the LBD, where lobe D2 moves to close

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around the ligand (Armstrong and Gouaux, 2000). On the other hand, antagonists stabilize an open LBD structure. Domain movements have either been calculated as a domain closure using DynDom (Hayward and Berendsen, 1998) (Table 2 and 3) or

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characterized by a two-dimensional order parameter (ξ1, ξ2; Fig. 8A), describing the

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large-scale conformational transitions of the LBDs (Roux and Lau, 2011).

All domain closures in Table 2 and 3 have been calculated relative to the second most open structure of GluK1-LBD (PDB code 3S2V, chain B). A full domain closure of 35-38° is seen in GluK1-LBD with (S)-glutamate. In agreement with partial agonist activity of kainate and domoate (Table 2), the GluK1-LBD complex is stabilized by kainate and domoate in a conformation that is more open than the conformation observed upon binding of glutamate: domain closure of 25-28° and 24-25° is seen for kainate and domoate, respectively. This more open conformation is caused by the

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ACCEPTED MANUSCRIPT presence of the isopropenyl group in kainate and the 5-carboxy-1-methyl-hexa-1,3dienyl moiety in domoate. All antagonists stabilize an open conformation of the LBD, varying from -19 to 26°. The antagonist (S)-2-amino-4-(2,3-dioxo-1,2,3,4tetrahydroquinoxalin-6-yl)butanoic acid shows a large variation in domain closures of

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the three molecules in the asymmetric unit of the crystal, varying from 19° further domain opening to 14° domain closure compared to the reference structure. This observation demonstrates that the GluK1-LBD is capable of undergoing major

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domain movements.

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Contradicting a relationship between domain closure and compound efficacy is the observation that dysiherbaine and analogues induce full to almost full domain closure regardless of agonist efficacy (Frydenvang et al., 2009; Unno et al., 2011). Also, full domain closure is seen for all GluK2 (33-36°) and GluK3 (35-36°) agonists

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crystallized, except kainate which at GluK2-LBD induces 29-31° closure and at GluK3-LBD 29-32° as well as domoate that at GluK2-LBD leads to a domain closure of 24-25°. Therefore, receptor activation is more complex than initially assumed.

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A plot of ξ1 versus ξ2 values for all agonists and antagonists are shown in Fig. 8B.

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Agonists lead to ξ1 values in the range 9.1-9.6 Å in GluK1-3, whereas the ξ2 values show a much larger variation (8.3-10.6 Å) compared to ξ1 values. It is a characteristic that antagonists induce larger ξ1 and ξ2 values in GluK1-LBD than agonists, with ξ1 values of 9.3-15.4 Å and ξ2 values in the range 10.5-17.8 Å (all chains included). From the plot in Fig. 8B there appears to be a linear correlation between the ξ1 and ξ2 values for antagonists.

4.4 Ions

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ACCEPTED MANUSCRIPT Activation of kainate receptors by glutamate requires the presence of both sodium and chloride ions (Bowie, 2010). In 2007, Plested and Mayer localized the binding site for chloride ions to the GluK1-LBD dimer interface and one year later they demonstrated that the sodium site was also located at the dimer interface (Plested et al., 2008). In

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this way, the ions stabilize the dimer (Fig. 9A). In the absence of sodium and chloride ions, dimer stability is reduced and desensitization rate increases (Table 4).

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The anion site is located 10 Å below the top surface of lobe D1, between four

intersubunit salt bridges: Arg775-Asp776 and Glu524-Lys531. The two basic residues

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Lys531 and Arg775 as well as two water molecules form contacts to the chloride ion (Fig. 9B). Two symmetrical cation binding sites were identified, formed by Glu524 and Asp528 (Fig. 9C). GluK1-LBD was analyzed with a range of cations (Table 4), showing that sodium ions bind with the best affinity and lead to the highest efficacy.

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The stoichiometry for binding of anions and cations was found to be 1:2. Veran et al. (2012) identified a zinc binding site in GluK3 based on mutation and electrophysiology data as well as modeling of a GluK3-LBD dimer. The site was

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localized at the bottom of the LBD dimer interface and involves Asp790 and His793 from one subunit and Asp761 from the other subunit of the GluK3-LBD. Thus, the

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LBD dimer interface of kainate receptors provides several sites for modulation by ions.

4.5 Dimer interface

In the structure of GluK1-LBD with glutamate (PDB code 2F36), 32 residues are involved in the formation of the dimer interface. Of these residues, 21 are conserved among all subunits (Table 5), suggesting a common role. In low-affinity kainate receptors, we only see variation at three positions: Tyr521, Ile770 and Gly789 in

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ACCEPTED MANUSCRIPT GluK1, which in GluK2 correspond to Tyr, Met and Gly and in GluK3 to His, Met and Asp. Some residues are unique between low-affinity and high-affinity kainate receptors: Val522, Lys696, Lys698, Ile699, Ser761, Ile780 and Glu788 in GluK1, which correspond to Glu, Asn, Arg, Tyr, Thr, Leu and Asn in the high-affinity kainate

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receptor subunits. These differences pinpoint residues of importance for obtaining characteristic features of low-affinity versus high-affinity kainate receptors at the

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LBD level and might form interesting sites for selective modulation.

Four different studies have been reported in which structures of GluK2-LBD were

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determined in order to address stability of the dimer interface (Table 6, Fig. 10). For example, Weston et al. (2006) attempted to build non-desensitizing kainate-subtype glutamate receptors by introducing an intermolecular disulfide cross-link (Y521C L783C), whereas Nayeem et al. (2011 and 2013) addressed the role of dimer

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conformation for gating and desensitization and were able to show that mutations can be introduced that abolish either cation or anion binding. Chaudhry et al. (2009) designed a range of dimer interface mutants, revealing an inverse correlation between

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dimer stability and the rate of desensitization. Despite similar structures, these

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mutants were shown to possess remarkably different functional properties.

5. Conclusion

The first structure of a kainate receptor was reported in 2005, seven years after publication of the crystal structure of a soluble construct of the ligand-binding domain of the AMPA-type receptor GluA2: the GluK1-LBD with (S)-glutamate bound. Since then extensive studies have been reported on kainate receptors, and today more than 80 crystal structures have been reported on soluble constructs of kainate receptors. However, most structural studies have been on the low-affinity kainate receptor

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ACCEPTED MANUSCRIPT subunits GluK1-3, and only structures of the N-terminal domain of the high-affinity kainate receptor GluK5 have been determined. The structures of wild-type and mutant kainate receptors, combined with functional data, have led to models for receptor activation and desensitization by agonists as well as inhibition by antagonists. Further,

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the structures have provided some understanding of subunit selectivity.

However, there are still many questions and challenges in front of us. How can small

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molecules modulate kainate receptors and where precisely are binding sites for

positive and negative allosteric modulators located in the kainate receptors? How can

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structures guide the design of highly selective agonists and antagonists? There is no doubt that it will be important to obtain structures of the kainate receptor subunits and domains that have so far escaped structure determination as well as structures of fulllength homomeric and heteromeric kainate receptors in multiple conformational states

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to fully understand kainate receptor function at the molecular level. Also, such studies might aid the design and development of future drugs for treatment of diseases and disorders in the brain.

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Acknowledgements

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The authors would like to acknowledge financial support for their work from GluTarget, The Lundbeck Foundation, The Novo Nordisk Foundation, Danscatt and BioStruct-X. Furthermore, we would like to acknowledge all the synchrotron facilities without which the present 84 crystal structures would not have been possible to analyze.

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ACCEPTED MANUSCRIPT References

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ACCEPTED MANUSCRIPT Figure legends

Fig. 1. Full-length GluK2 structure determined by EM to 7.6 Å resolution (PDB code 4UQQ). (A) Surface representation of the structure with the four subunits colored

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differently. (B) The NTD dimer in the desensitized full-length GluK2 is similar to the dimer of a soluble NTD construct (PDB code 3QLT). (C) The LBD dimer is very

different in the full-length GluK2 compared to a soluble LBD construct (PDB code

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2XXR).

Fig. 2. The GluK2-NTD crystallizes as a dimer (PDB code 3QLT). One NTD subunit

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is shown in green and one in blue, with lobe R1 in dark colors and lobe R2 in light colors. Carbohydrate moieties are shown in yellow stick representation.

Fig. 3. LBDs of kainate receptors. (A) The LBD is comprised of a clamshell structure,

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composed of two lobes D1 (light cyan) and D2 (cyan). Shown is a cartoon representation of the GluK1-LBD in complex with (S)-glutamate (orange) (PDB code 2F36). (B) An overlay of the LBD structures of GluK1-3 in complex with (S)-

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glutamate (PDB codes 2XXR for GluK2 and 4MH5 for GluK3). The structures have

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been superimposed on lobe D1 residues. GluK2 is shown in blue and GluK3 in green.

Fig. 4. Zoom on the binding site for (S)-glutamate. (A) Hydrogen-bonding interactions (black stippled lines) between (S)-glutamate (orange) and GluK1 binding site residues (cyan) and water molecules (red spheres). (B) Three binding site differences in GluK1-3. Glu441, which is conserved in all subunits, is included to show the important D1-D2 interlobe contact. Numbering is for GluK1 and PDB codes as in Fig. 3.

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ACCEPTED MANUSCRIPT Fig. 5. Zoom on interactions of kainate, domoate and dysiherbaine in GluK1-LBD. (A) Hydrogen-bonding interactions (black stippled lines) between kainate (orange) and GluK1 binding site residues (cyan) and water molecules (red spheres). The 4isopropenyl group of kainate weakens the Glu441-Ser721 interlobe contact (orange

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stippled line). PDB code 4E0X. (B) Hydrogen-bonding interactions between domoate (orange) and GluK1 binding site residues and water molecules. PDB code 2PBW. (C) Binding of dysiherbaine in GluK1-LBD. A short interlobe contact is seen. PDB code

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3GBA.

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Fig. 6. Binding site water molecules. (A) Seven binding site water molecules (W1-7) are located within 5 Å of glutamate in GluK1-LBD. Agonists can displace water molecules W1, W2, W5 and W7 (beige), whereas W3, W4 and W6 (red) are present in crystal structures with agonists. PDB code 2F36.

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Fig. 7. Binding of antagonists in GluK1-LBD. (A) Binding mode of (S)-1-(2'-amino2'-carboxyethyl)-3-[(2-carboxythien-3-yl)methyl]thieno[3,4-d]pyrimidin-2,4-dione (orange, PDB code 3S2V). Four residues are shown: Arg523 as a lobe D1 anchor

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residue, Glu738 forming a salt bridge with the antagonist as well as Glu441 in D1 and

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Ser721 in D2, which form an interlobe contact in agonist structures. These four residues are displayed as cyan sticks. (B) Comparison of the binding mode of UBP310 (black, PDB code 2OJT) and UBP315 (orange, PDB code 2QS1). The structure of GluK1-LBD with UBP310 is shown as dark grey cartoon and with Glu738 in black, whereas the structure with UBP315 is shown as light grey cartoon and with residues in cyan. (C) Binding mode of (3S,4aS,6S,8aR)-6-[3-chloro-2-(1Htetrazol- 5-yl)phenoxy]deca-hydroisoquinoline-3-carboxylic acid (orange, PDB code 4MF3).

30

ACCEPTED MANUSCRIPT Fig. 8. Domain movements characterized by a two-dimensional order parameter (ξ1, ξ2). (A) Definition of ξ values (backbone atoms only): GluK1: ξ1 is the distance (black line) from the center of mass of Leu517, Thr518 and Ile519 (red) to the center of mass of Ser689 and Thr690 (orange); ξ2: Leu440, Glu441 and Glu442 (cyan) to Ser721 and

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Asp722 (green). GluK2: ξ1: Leu517, Ala518 and Ile519 to Ala689 andThr690; ξ2:

Leu439, Glu440 and Glu441 to Asn721 and Glu722. GluK3: ξ1: Leu519, Thr520 and Ile521 to Ala691 and Thr692; ξ2: Leu442, Glu443 and Glu444 to Asn722 and Glu723.

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(B) Plot of ξ1versus ξ2. Agonists are represented by a circle and antagonists by a

rectangle. Color coding: GluK1 cyan, GluK2 blue and GluK3 green. Numbering of

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ligands is according to Tables 2 and 3. The values for structures from rat and for chain A are shown. Where more than one structure have been determined with a given ligand the structure was selected based on resolution.

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Fig. 9. Anion and cation binding sites at the dimer interface of GluK1-LBD. (A) Localization of anion (green) and cation (violet) binding sites. Two different views are shown, rotated by 90°. (B) Zoom on the anion binding site. (C) Zoom on the

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cation binding site. PDB code 2C32.

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Fig. 10. Dimer interface mutations in GluK2-LBD (PDB code 2XXR). The 13 residues that have been mutated (Table 6) are shown in blue sticks (chain A) and light blue sticks (chain B) representation. Residues in chain A have been labeled.

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ACCEPTED MANUSCRIPT

PDB ID

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Table 1. Crystal structures of kainate receptor N-terminal domains.

Resolution (Å)

Source

Reference

2.70 2.90 2.99

Rat Rat Rat

Kumar et al., 2009 Kumar et al., 2009 Kumar et al., 2011

2.75

Rat

Kumar and Mayer, 2010

1.40 1.68

Rat Rat

Kumar and Mayer, 2010 Kumar and Mayer, 2010

2.91 3.94

Rat Rat

Kumar et al., 2011 Kumar et al., 2011

GluK3 3OLZ GluK5 3OM0 3OM1

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3QLU 3QLV

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GluK2/GluK5

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3H6G 3H6H 3QLT

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GluK2

1

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Resolution (Å)

Source

D1-D2 closure (deg)

2WKY 8-deoxyneodysiherbaine A (2) 3FVK 9-deoxyneodysiherbaine A (3) 3FVN Domoate (4)

2.2

Rat

26.7/26.6

1.5

Human

37.6/38.3

1.5

Human

37.8/38.4

2PBW Dysiherbaine (5)

2.5

Rat

24.4/24.5

2ZNT

1.6

Human

GluK1

AC C

EP

4-AHCP (1)

Binding affinity at LBD (nM)

29.7

Binding affinity at fulllength (nM)

Potency (nM)

Efficacy (%)

Reference

2.6

580a

147a

Clausen et al., 2009

1.5b

0.24b

Unno et al., 2011

169b

151b

Unno et al., 2011

1.1

13,000c

36c

Hald et al., 2007

0.48d

210d

100d

Unno et al., 2011

TE D

PDB ID

M AN U

SC

RI PT

Table 2. Crystal structures of kainate receptor ligand-binding domains in complex with agonists.

5.6

2

ACCEPTED MANUSCRIPT

3FV1 3GBA

1.5 1.35

Human Rat

36.4/38.0 36.5/36.3/ 35.6/35.7

Unno et al., 2011 Frydenvang et al., 2009

8-epi-neodysiherbaine A (6) 3FVO

1.5

Human

37.4/38.3

1TXF 1YCJ 2F36

2.1 1.95 2.11

Rat Rat Rat

2ZNS 3FUZ Kainate (8)

2.0 1.65

Human Human

35.1 36.0/37.7 35.5/36.5/ 36.6/35.2 34.7 37.2/38.6

3C31 3C32 3C33 3C34 3C35 3C36 4E0X

1.49 1.72 1.72 1.82 1.97 1.68 2.0

Rat Rat Rat Rat Rat Rat Rat

25.6/25.4 26.1/26.0 25.9/26.6 26.2/26.3 25.8/25.9 25.9/26.0 27.3/27.6

Neodysiherbaine A (9) 2ZNU 3FV2 MSVIII-19 (10)

1.8 1.5

Human Human

30.0 38.3/36.5

7.7h

Unno et al., 2011 Unno et al., 2011

3FVG 3GBB

1.5 2.1

Human Rat

37.6/38.1 33.6/35.7

128 161

Unno et al., 2011 Frydenvang et al., 2009

RI PT

0.85

34b

Unno et al., 2011

140e

917,000c

100c

M AN U

57

SC

Glutamate (7)

Unno et al., 2011 Unno et al., 2011

TE D

290

AC C

EP

84f

126

76g

51,000

3,600

Mayer, 2005 Naur et al., 2005 Mayer et al., 2006

36

13

Plested et al., 2008 Plested et al., 2008 Plested et al., 2008 Plested et al., 2008 Plested et al., 2008 Plested et al., 2008 Venskutonytė et al., 2012a

3

ACCEPTED MANUSCRIPT

GluK2 Domoate (4) Rat

24.3/24.8/ 25.0/24.2/ 24.4/24.5

3.8

20

290i

1S50 1S7Y 3G3F 2XXR GluAzo (11)

1.65 1.75 1.38 1.6

Rat Rat Rat Rat

36.1 35.8/35.7 34.3/35.8 33.0/33.1

1,400

252k

108,000l

4H8I

2

Rat

29.1/ 10.3(MES)

1TT1 2XXT (2S,4R)-4Methylglutamate (12) 1SD3 Neodysiherbaine A (9) 3QXM Quisqualate (13)

1.93 1.9

Rat Rat

31.2/31.0 30.2/29.2

1.8

Rat

35.4/35.3

1.65

Human

34.2/34.1

1S9T GluK3

1.8

Rat

35.8/36.0

3S9E

1.6

Rat

3U93 3U94

1.88 1.96

Rat Rat

36

EP

AC C 35.9/35.9

35.1/35.1 35.2/35.3/

13g

TE D

65

M AN U

Kainate (8)

Glutamate (7)

Nanao et al., 2005

100l

Mayer, 2005 Mayer, 2005 Chaudhry et al., 2009 Nayeem et al., 2011

SC

Glutamate (7)

15j

RI PT

3.11

1YAE

253

17g

Reiter et al., 2013

1,100i

39j

Mayer, 2005 Nayeem et al., 2011

1,550l

72l

Mayer, 2005

33h 134g

Unno et al., 2011 50,000a

101a

Mayer, 2005

Venskutonytė et al., 2011a Veran et al., 2012 Veran et al., 2012

4

ACCEPTED MANUSCRIPT

Rat

4G8N Kainate (8)

2.3

Rat

36.0

3U92 4E0W

1.9 2.35

Rat Rat

32.3/29.4 31.8

(2S,4R)-4-(3Methoxy-3oxopropyl)glutamic acid (15) 4NWC

2.01

Rat

35.2

(2S,4R)-4-(3Methylamino-3oxopropyl)glutamic acid (16) 4NWD

2.6

Rat

34.9

4MH5 (replaces 3S9E)

7,430

494g

4,180

325

9,030,00 0l

G8M (14)

27l

16l

SC 1,010g

3,960

567

Venskutonytė et al., 2011a Juknaitė et al., 2012

M AN U

1,740

TE D

ZA302 (17)

33g

100

RI PT

1.65

36.5/35.4 35.9

Veran et al., 2012 Venskutonytė et al., 2012a

Venskutonytė et al., 2014

Venskutonytė et al., 2014

AC C

EP

4IGR 2.65 Rat 34.7 900 123 Assaf et al., 2013 a Strange et al., 2006. bLash et al., 2008. cVenskutonyté et al., 2012a. dSakai et al., 2001. eAssaf et al., 2013. fMayer 2005. gSagot et al., 2008. hSanders et al., 2005. iAlt et al., 2004. jFay et al., 2009. kNanao et al., 2005. lVenskutonyté et al., 2011b.

5

ACCEPTED MANUSCRIPT

Source

2.5

Rat

3S2V

Binding affinity at LBD (µM)

Binding affinity at full-length (µM)

KB (µM)

Reference

0.16

0.087

Venskutonytė et al., 2011b

EP

(S)-1-(2'-Amino-2'carboxyethyl)-3-[(2carboxythien-3yl)methyl]thieno[3,4d]pyrimidin-2,4-dione (21)

D1-D2 closure (deg)

TE D

Resolution (Å)

AC C

PDB ID

M AN U

SC

RI PT

Table 3. Crystal structures of GluK1 ligand-binding domains in complex with antagonists.

10.5/0 Reference molecule

8.6a

6

(S)-2-Amino-3-(2-(2carboxyethyl)-5chloro-4nitrophenyl)propionic acid (22) 2

Rat

23.7/25.7

24b

4QF9

2.28

Rat

13.8/10.3/ -19.3

37

1VSO CNG10111 (25)

1.85

Rat

11.4

19

4YMB LY466195 (26)

1.93

Rat

20.7/19.6

2QS4

1.58

Rat

n.d.d/12.5/12. 7/13.0

3

Human

11.1/12.3

1.87

Rat

7.0/7.2

3.8b

Venskutonytė et al., 2012b

0.038

M AN U 16

2.2

23c

0.62 0.05e

Demmer et al., 2015

Hald et al., 2007 Krogsgaard-Larsen et al., 2015

0.024e

Alushin et al., 2011

EP

AC C

2F35 UBP310 (29)

TE D

(S)-ATPO (24)

(3S,4aS,6S,8aR)-6-[3chloro-2-(1H-tetrazol5-yl)phenoxy]decahydroisoquinoline-3carboxylic acid (27) 4MF3 UBP302 (28)

3.0b

SC

4DLD (S)-2-Amino-4-(2,3dioxo-1,2,3,4tetrahydroquinoxalin6-yl)butanoic acid (23)

RI PT

ACCEPTED MANUSCRIPT

0.2 3.9

0.40f

Martinez-Perez et al., 2013 106f

Mayer et al., 2006

7

ACCEPTED MANUSCRIPT

2F34 2OJT UBP315 (30)

1.74 1.95

Rat Rat

7.2/7.1 6.7/6.7

0.13

0.022g

0.010h

2QS1 UBP316 (31)

1.8

Rat

10.5/10.5

0.033

0.010h

2.8h

Alushin et al., 2011

2QS3 UBP318 (32)

1.76

Rat

7.1/6.8

0.012h

0.0014

Dargan et al., 2009

2QS2

1.8

Rat

7.0/7.1

RI PT

43h

Alushin et al., 2011

Pickering, D. S., personal data. bRacemate. cMøller et al., 1999. dNot determined. eWeiss et al., 2006. fMore et al., 2004. gKrogsgaard-Larsen et al., 2015. hDolman et al., 2007.

AC C

EP

TE D

M AN U

a

SC

0.025h

0.19

Mayer et al., 2006 Plested and Mayer, 2007

8

ACCEPTED MANUSCRIPT

kdesa (s-1)

Resolution (Å)

Ligand

Reference

1.95

UBP310

3C36 Cesium

1.68

Kainate

1200

Plested et al., 2008

3C35 Lithium

1.97

Kainate

1900

Plested et al., 2008

3C31 Potassium

1.49

Kainate

180

Plested et al., 2008

3C33 Rubidium

1.72

Kainate

780

Plested et al., 2008

3C34 Sodium

1.82

Kainate

1300

Plested et al., 2008

ANIONS Bromide 2OJT CATIONSb

Plested and Mayer, 2007

TE D

M AN U

Ammonium

SC

PDB ID

RI PT

Table 4. Cation and anion binding sites at the dimer interface of the rat GluK1 ligand-binding domain.

AC C

EP

3C32 1.72 Kainate 160 Plested et al., 2008 a kdes: Desensitization constant. b The cation containing structures also have a chloride ion at the dimer interface.

9

ACCEPTED MANUSCRIPT

RI PT

Table 5. Residues involved in LBD dimer interaction in kainate receptors.

GluK1 numbering 519 520 521 522

524 525

528 529 530 531 532

535

692 693

696 697 698 699

GluK1

Ile

Thr Tyr

Val

Glu Lys

Asp Phe Ser Lys

Pro

Thr

Thr Phe

Lys

Ser Lys

GluK2

Ile

Thr Tyr

Val

Glu Lys

Asp Phe Ser Lys

Pro

Thr

Thr Phe

Lys

Ser Lys

GluK3

Ile

Thr His

Val

Glu Lys

Asp Phe Ser Lys

Pro

Thr

Thr Phe

Lys

Ser Lys

GluK4

Ile

Thr Ala

Glu

Glu Lys

Asp Phe Ser Lys

Pro

Thr

Thr Phe

Asn Ser Arg Tyr

GluK5

Ile

Thr Ala

Glu

Glu Lys

Asp Phe Ser Lys

Pro

Thr

Thr Phe

Asn Ser Arg Tyr

775 776

779 780

783 784

786 787 788 789

Ile

Arg Asp

Thr Ile

Leu Gln

Gln Glu Glu Gly

GluK2

Asp Ser Lys

Met

Arg Asp

Thr Ile

Leu Gln

Gln Glu Glu Gly

GluK3

Asp Ser Lys

Met

Arg Asp

Thr Ile

Leu Gln

Gln Glu Glu Asp

GluK4

Asp Thr Lys

Val

Arg Asp

Asp Leu

Leu Gln

Gln Glu Asn Asn

GluK5

Asp Thr Lys

Leu

Arg Asp

Thr Leu

Leu Gln

Gln Glu Asn Asn

Ile

SC

770

Asp Ser Lys

Ile

AC C

EP

TE D

M AN U

GluK1 numbering 760 761 762 GluK1

Ile

10

ACCEPTED MANUSCRIPT

kdes (s-1)

Reference

Glutamate Glutamate Glutamate Kainate Glutamate Glutamate Kainate Glutamate Glutamate Glutamate Glutamate

1.96 2.25 1.50 1.70 2.30 2.10 3.00 1.3 1.5 1.37 1.32

108 68a

Weston et al., 2006 Weston et al., 2006 Nayeem et al., 2011 Nayeem et al., 2011 Nayeem et al., 2011 Nayeem et al., 2011 Nayeem et al., 2011 Chaudhry et al., 2009 Chaudhry et al., 2009 Chaudhry et al., 2009 Chaudhry et al., 2009

Glutamate

1.24

Glutamate Kainate Glutamate Kainate Glutamate Kainate

1.75 3.40 2.50 2.55 1.90 1.65

M AN U

400a

SC

Resolution (Å)

36 5.0 13 1.5

TE D

K525E I780L Q784K E788Q Y521C L783C M770K M770K D776K D776K D776K K696R K696R I780L Q784K I473H K525E I780L Q784K I473H K525E K696R I780L Q784K 3G3K I473H K525E K696R I780L Q784K E788Q 4BDL K531A 4BDM K531A 4BDN K531A T779G 4BDO K531A T779G 4BDQ R775A 4BDR R775A a Dawe et al., 2013

Ligand

EP

2I0B 2I0C 2XXU 2XXV 2XXW 2XXX 2XXY 3G3G 3G3H 3G3I 3G3J

Mutation

AC C

PDB ID

RI PT

Table 6. Crystal structures where mutations have been introduced into the dimer interface in the rat GluK2 ligand-binding domain.

6.7

Chaudhry et al., 2009

120 11 5.3 4.5 99 61

Nayeem et al., 2013 Nayeem et al., 2013 Nayeem et al., 2013 Nayeem et al., 2013 Nayeem et al., 2013 Nayeem et al., 2013

11

ACCEPTED MANUSCRIPT Figure 1

A

M AN U

LBD

SC

RI PT

NTD

TMD

TE D

B

Soluble NTD

AC C

EP

EM NTD

C

 

EM LBD

Soluble LBD

1  

ACCEPTED MANUSCRIPT Figure 2

SC

RI PT

R1

AC C

EP

TE D

M AN U

R2

 

2  

ACCEPTED MANUSCRIPT Figure 3

A

B

SC

RI PT

D1

AC C

EP

TE D

M AN U

D2

 

3  

ACCEPTED MANUSCRIPT Figure 4

A L517 R523

RI PT

P516 T518 W1 E738

W4

T690

B

M AN U

W2

SC

S689

W3

E441

S689

TE D

T518

AC C

EP

S721

 

4  

ACCEPTED MANUSCRIPT Figure 5

A

L517

T518

E441

R523 S689 E738

4.0 Å S721

SC

W2

M AN U

W4 T690 W3

B

RI PT

P516

P516

T518 Y489

R523

E441

E738 S689

4.0 Å

S721

TE D

W2

W4

T690 W3

L517

T518

AC C

R523

P516

EP

C

E441

E738

S689

2.9 Å S721

W4

 

T690 W3

5  

ACCEPTED MANUSCRIPT Figure 6

W6

W5 W2

W4

SC

W7

RI PT

W1

AC C

EP

TE D

M AN U

W3

 

6  

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Figure 7

 

7  

ACCEPTED MANUSCRIPT Figure 8

M AN U

SC

RI PT

A

AC C

EP

TE D

B

 

8  

ACCEPTED MANUSCRIPT Figure 9

K531(B)

E524

D528

AC C

EP

TE D

K531(A)

C

SC

R775(B) R775(A)

M AN U

B

RI PT

A

 

9  

ACCEPTED MANUSCRIPT Figure 10

K696 K525

M770 R775 K531

D776

M AN U

T779

SC

RI PT

Y521

L783

I780

I473 Q784

AC C

EP

TE D

E788

 

10