Cysteine mutagenesis and homology modeling of the ligand-binding site of akainate-binding protein

Glutamate receptors comprise the most abundant group of neurotransmitter re ceptors in the vertebrate central nervous system. Cysteine mutagenesis in c ombination with homology modeling has been used to study the determinants o f kainate binding in a glutamate receptor subtype, a low molecular weight g oldfish kainate-binding protein, GFKAR beta. A construct of GFKAR beta with no cysteines in the extracellular domain was produced, and single cysteine residues were introduced at selected positions. N Ethylmaleimide or deriva tized methanethiosulfonate reagents (neutral or charged) were used to modif y the introduced cysteines covalently, and the effect on [H-3]kainate bindi ng was determined. In addition, cysteine mutants of GFKAR beta transiently expressed in HEK293 cells were labeled with a membrane-impermeable biotinyl ating reagent followed by precipitation with streptavidin beads and specifi c detection of GFKAR beta by Western blot analysis. The results are consist ent with the proposal that the energy driving kainate binding is contribute d 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 u pon ligand binding in a manner analogous to that seen in bacterial amino ac id-binding proteins.