Binding site and ligand flexibility revealed by high resolution crystal structures of GluK1 competitive antagonists

Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, NICHD, NIH, DHHS, Bethesda, MD 20892, USA.
Neuropharmacology (Impact Factor: 5.11). 01/2011; 60(1):126-34. DOI: 10.1016/j.neuropharm.2010.06.002
Source: PubMed


The availability of crystal structures for the ligand binding domains of ionotropic glutamate receptors, combined with their key role in synaptic function in the normal and diseased brain, offers a unique selection of targets for pharmaceutical research compared to other drug targets for which the atomic structure of the ligand binding site is not known. Currently only a few antagonist structures have been solved, and these reveal ligand specific conformational changes that hinder rational drug design. Here we report high resolution crystal structures for three kainate receptor GluK1 antagonist complexes which reveal new and unexpected modes of binding, highlighting the continued need for experimentally determined receptor-ligand complexes.

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    • "Whereas molecular modeling of ligand-KAR interactions has been used to design more potent and subunit-selective antagonists (Dolman et al., 2007; Dargan et al., 2009; Jane et al., 2009), the involvement of individual amino acid residues in the subunit-selective binding of antagonists to KARs has not been established experimentally. In addition, a complication in the drug design process is that the degree of opening of the LBD of GluK1, the conformation of flexible side chains of residues in the LBD, and the position of water molecules in the binding site can vary with the structure of the interacting ligand (Mayer et al., 2006; Alushin et al., 2010), and so information regarding the involvement of nonconserved residues in ligand binding from site-directed mutagenesis studies would facilitate the design of KAR subunit-selective antagonists. Site-directed mutagenesis of key amino acid residues likely to be involved in ligand binding is often used to confirm predictions made by molecular modeling. "
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    ABSTRACT: Kainate receptors (KARs) modulate synaptic transmission and plasticity, and their dysfunction has been linked to several disease states such as epilepsy and chronic pain. KARs are tetramers formed from five different subunits. GluK1-3 are low affinity kainate binding subunits, whereas GluK4/5 bind kainate with high affinity. A number of these subunits can be present in any given cell type, and different combinations of subunits confer different properties to KARs. Here we report the characterization of a new GluK1 subunit-selective radiolabeled antagonist (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione ([(3)H]UBP310) using human recombinant KARs. [(3)H]UBP310 binds to GluK1 with low nanomolar affinity (K(D) = 21 ± 7 nM) but shows no specific binding to GluK2. However, [(3)H]UBP310 also binds to GluK3 (K(D) = 0.65 ± 0.19 μM) but with ~30-fold lower affinity than that observed for GluK1. Competition [(3)H]UBP310 binding experiments on GluK1 revealed the same rank order of affinity of known GluK1-selective ligands as reported previously in functional assays. Nonconserved residues in GluK1-3 adjudged in modeling studies to be important in determining the GluK1 selectivity of UBP310 were point-mutated to switch residues between subunits. None of the mutations altered the expression or trafficking of KAR subunits. Whereas GluK1-T503A mutation diminished [(3)H]UBP310 binding, GluK2-A487T mutation rescued it. Likewise, whereas GluK1-N705S/S706N mutation decreased, GluK3-N691S mutation increased [(3)H]UBP310 binding activity. These data show that Ala487 in GluK2 and Asn691 in GluK3 are important determinants in reducing the affinity of UBP310 for these subunits. Insights from these modeling and point mutation studies will aid the development of new subunit-selective KAR antagonists.
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