Binding site and interlobe interactions of the ionotropic glutamate receptor GluK3 ligand binding domain revealed by high resolution crystal structure in complex with (S)-glutamate Raminta Venskutonyte ˙ a,b , Karla Frydenvang a , Michael Gajhede a , Lennart Bunch a , Darryl S. Pickering b , Jette S. Kastrup a,⇑ a Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark b Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark article info Article history: Received 16 June 2011 Received in revised form 24 August 2011 Accepted 25 August 2011 Available online 1 September 2011 Keywords: Ionotropic glutamate receptor X-ray crystallography Ligand binding site Receptor ligand interactions Dimerization abstract Ionotropic glutamate receptors (iGluRs) are involved in excitatory signal transmission throughout the central nervous system and their malfunction is associated with various health disorders. GluK3 is a sub- unit of iGluRs, belonging to the subfamily of kainate receptors (GluK1–5). Several crystal structures of GluK1 and GluK2 ligand binding domains have been determined in complex with agonists and antago- nists. However, little is known about the molecular mechanisms underlying GluK3 ligand binding prop- erties and no compounds displaying reasonable selectivity towards GluK3 are available today. Here, we present the first X-ray crystal structure of the ligand binding domain of GluK3 in complex with gluta- mate, determined to 1.6 Å resolution. The structure reveals a conserved glutamate binding mode, char- acteristic for iGluRs, and a water molecule network in the glutamate binding site similar to that seen in GluK1. In GluK3, a slightly lower degree of domain closure around glutamate is observed compared to most other kainate receptor structures with glutamate. The volume of the GluK3 glutamate binding cavity was found to be of intermediate size between those of GluK1 and GluK2. The residues in GluK3 contributing to the subfamily differences in the binding sites are primarily: Thr520, Ala691, Asn722, Leu736 and Thr742. The GluK3 ligand binding domain seems to be less stabilized through interlobe inter- actions than GluK1 and this may contribute to the faster desensitization kinetics of GluK3. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Ionotropic glutamate receptors (iGluRs) are tetrameric ligand gated ion channels, widely spread throughout the central nervous system where they play a crucial role in fast excitatory signal transmission. iGluRs are divided into three subfamilies on the basis of ligand selectivity and protein sequence identity: 2-amino-3-(3- hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), N-methyl- D-aspartic acid (NMDA) and kainate receptors (KARs). GluK3 belongs to the kainate receptor subfamily (GluK1–5) (Traynelis et al., 2010). It has been shown that GluK1–3 may form functional homomeric or heteromeric receptors whereas GluK4 and GluK5 only form functional receptors in combination with other KAR subunits (Alt et al., 2004; Cui and Mayer, 1999). KARs have been linked to various neurological conditions including pain, epilepsy and migraine. Further, KARs differ from the remaining iGluRs as they mostly have a modulatory function in synaptic transmission, a feature that makes them an attractive therapeutic target (Contractor et al., 2011; Jane et al., 2009). GluK3 is differentially expressed in various brain regions; how- ever, the physiological function of this subunit is not clear. On a gene level, GRIK3 was associated with recurrent major depressive disorder (Schiffer and Heinemann, 2007), thus making it a poten- tial drug target. Furthermore, evidence exist that GluK3 functions as a presynaptic receptor in MF-CA3 synapses as a heteromer to- gether with GluK2, where it facilitates synaptic transmission. It was shown that long term potentiation is impaired in GluK3/ mice (Pinheiro et al., 2007). Interestingly, GluK3 displays a very low potency to glutamate compared to other iGluRs, with EC 50 val- ues being around 10 mM, which suggests a specific physiological function (Perrais et al., 2009; Pinheiro et al., 2007; Schiffer et al., 1997). iGluRs consist of an intracellular carboxy-terminal domain (CTD), a transmembrane domain (TMD), an extracellular ligand 1047-8477/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2011.08.014 Abbreviations: AMPA, 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid; ATD, amino-terminal domain; CTD, carboxy-terminal domain; iGluRs, iono- tropic glutamate receptors; IPTG, isopropyl-b-D-thiogalactopyranoside; KARs, kai- nate receptors; LBD, ligand binding domain; NMDA, N-methyl-D-aspartic acid; TMD, transmembrane domain. ⇑ Corresponding author. Fax: +45 3533 6001. E-mail address: jsk@farma.ku.dk (J.S. Kastrup). Journal of Structural Biology 176 (2011) 307–314 Contents lists available at SciVerse ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi