Cysteine Mutagenesis and Homology Modeling of the Ligand-binding Site of a Kainate-binding Protein

Abstract

Glutamate receptors comprise the most abundant group of neurotransmitter receptors in the vertebrate central nervous system. Cysteine mutagenesis in combination with homology modeling has been used to study the determinants of kainate binding in a glutamate receptor subtype, a low molecular weight goldfish kainate-binding protein, GFKAR␤. A construct of GFKAR␤ with no cysteines in the extracellular domain was produced, and single cysteine residues were introduced at selected positions. N-Ethylmaleimide or derivatized methanethiosulfonate reagents (neutral or charged) were used to modify the introduced cysteines covalently, and the effect on [ 3 H]kainate binding was determined. In addition, cysteine mutants of GFKAR␤ transiently expressed in HEK293 cells were labeled with a membrane-impermeable biotinylating reagent followed by precipitation with streptavidin beads and specific detection of GFKAR␤ by Western blot analysis. The results are consistent with the proposal that the energy driving kainate binding is contributed both from residues within the binding site and from interactions between two regions (i.e. two lobes) of the protein that are brought into contact upon ligand binding in a manner analogous to that seen in bacterial amino acid-binding proteins. Glutamate receptors (GluRs)1 are the major excitatory receptors in the vertebrate central nervous system and have been implicated in a number of normal and pathologic processes including synaptic plasticity, epilepsy, and ischemic cell death (1). GluRs consist of two large superfamilies of membranebound receptors: (i) metabotropic receptors, which are linked by G proteins to second messengers such as phospholipase C, and (ii) ionotropic receptors (iGluRs), which are ligand-gated ion channels. The iGluRs have been subdivided based upon agonist sensitivity and sequence homology into three subfamilies (2, 3): (i) N-methyl-D-aspartate receptors (R1-3), (ii) ␣-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors (GluR1-4), and (iii) kainate receptors (GluR5-7, KA1-2, and kainate-binding proteins from nonmammalian vertebrates). For all these subtypes, some uncertainty exists over the subunit stoichiometry; each receptor is thought to be composed of either four (4 -6) or five (7) subunits that form functional ion channels in the cell membrane.iGluRs have a distinct modular structure (Fig. 1A and Ref. 8). This consists of (i) one (S1S2, also known as LAOBP-like) or two (both S1S2 and leucine, isoleucine, valine-binding proteinlike) domains that are homologous to bacterial amino acidbinding proteins (9, 10), (ii) a channel domain that has some similarities to cyclic nucleotide-gated and potassium channels (8, 11), and (iii) a C-terminal domain that mediates regulation by phosphorylation (12) and binds intracellular proteins (13-15). In particular, the agonist and antagonist-binding domain (S1S2 domain; Refs. 8, 10, and 16) consists of a portion of the N terminus of the protein before the first transmembrane segment (M1) and a large portion of the p...

Influence of a threonine residue in the S2 ligand binding domain in determining agonist potency and deactivation rate of recombinant NR1a/NR2D NMDA receptors

“…A number of structure–function studies have identified the residues in the S1 and S2 domains of the glutamate receptor subunits that control glutamate binding (Kuusinen et al 1995; Paas et al 1996; Laube et al 1997; Anson et al 1998, 2000; Wo et al 1999). These findings have correlated well with evidence obtained from the analysis of the crystal structure of the GluR2 S1S2 protein fragment (Armstrong et al 1998; Armstrong & Gouaux, 2000).…”

Structural Features of the Glutamate Binding Site in Recombinant NR1/NR2A N-Methyl-D-aspartate Receptors Determined by Site-Directed Mutagenesis and Molecular Modeling

“…Several structure-function studies of ionotropic glutamate receptors and related kainate binding proteins have identified residues located in the S1 and S2 binding domains that, when mutated, lead to a reduction in the ability of the ligand to remain bound to its binding site (Kuryatov et al, 1994;Kuusinen et al, 1995;Paas et al, 1996;Laube et al, 1997Laube et al, , 2004Anson et al, 1998Anson et al, , 2000Wo et al, 1999;Kalbaugh et al, 2004; for review, see Erreger et al, 2004). This study is the first to describe systematically the effects of mutating each of the proposed contact residues in the ligand binding Fig.…”

Glutamate Receptor Trafficking: Endoplasmic Reticulum Quality Control Involves Ligand Binding and Receptor Function

“…Various alterations in side chain polarity, charge, or length of the ligand-binding-site residues impact radioligand binding to the KA-binding protein (Paas et al, 1996;Wo et al, 1999) and AMPA receptors (Kawamoto et al, 1997;Lampinen et al, 1998Lampinen et al, , 2002Abele et al, 2000;Jouppila et al, 2002). In the S1 domain, the conservative GluR4-R507K mutation eliminated highaffinity antagonist binding , and the equivalent GluR6-R523K mutation reportedly increased the EC 50 value for glutamate activation by Ͼ50-fold.…”