Probing the ligand binding domain of the GluR2 receptor by proteolysis anddeletion mutagenesis defines domain boundaries and yields a crystallizableconstruct

Ionotropic glutamate receptors constitute an important family of ligand-gat ed ion channels for which there is little biochemical or structural data. H ere we probe the domain structure and boundaries of the ligand binding doma in of the AMPA-sensitive GluR2 receptor by limited proteolysis and deletion mutagenesis. To identify the proteolytic fragments, Maldi mass spectrometr y and N-terminal amino acid sequencing were employed. Trypsin digestion of HS1S2 (Chen GQ, Gouaux E. 1997. Proc Natl Acad Sci USA 94:13431-13436) in t he presence and absence of glutamate showed that the ligand stabilized the S1 and S2 fragments against complete digestion. Using limited proteolysis a nd multiple sequence alignments of glutamate receptors as guides, nine cons tructs were made, folded, and screened for ligand binding activity. From th is screen, the S1S2I construct proved to be trypsin- and chymotrypsin-resis tant, stable to storage at 4 degrees C, and amenable to three-dimensional c rystal formation. The HS1S2I variant was readily prepared on a large scale, the His tag was easily removed by trypsin, and crystals were produced that diffracted to beyond 1.5 Angstrom resolution. These experiments, for the f irst time, pave the way to economical overproduction of the ligand binding domains of glutamate receptors and more accurately map the boundaries of th e ligand binding domain.