Probing the ligand binding domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct

Abstract

Ionotropic glutamate receptors constitute an important family of ligand-gated ion channels for which there is little biochemical or structural data. Here we probe the domain structure and boundaries of the ligand binding domain of the AMPA-sensitive GluR2 receptor by limited proteolysis and deletion mutagenesis. To identify the proteolytic fragments, Maldi mass spectrometry and N-terminal amino acid sequencing were employed. Trypsin digestion of HSlS2 (Chen GQ, Gouaux E. 1997. Proc NarZAcad Sci USA 94:13431-13436) in the presence and absence of glutamate showed that the ligand stabilized the SI and S2 fragments against complete digestion. Using limited proteolysis and multiple sequence alignments of glutamate receptors as guides, nine constructs were made, folded, and screened for ligand binding activity. From this screen, the SlS2I construct proved to be trypsin-and chymotrypsin-resistant, stable to storage at 4 "C, and amenable to three-dimensional crystal formation. The HS 1S2I 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 8, resolution. These experiments, for the first time, pave the way to economical overproduction of the ligand binding domains of glutamate receptors and more accurately map the boundaries of the ligand binding domain.

Solution X-ray Scattering Evidence for Agonist- and Antagonist-induced Modulation of Cleft Closure in a Glutamate Receptor Ligand-binding Domain

“…The full-length GluR4 LBD construct that had been used in previous solution scattering experiments ("S1S2-D") was generated using the baculovirus expression system and purified as previously described (24). Finally, a full-length GluR2 LBD ("S1S2L") was also refolded from bacterially expressed protein (23). S1S2L consisted of residues Ser 383 -Glu 524 (S1), a GTDGN linker, and Glu 627 -Asn 791 (S2), corresponding approximately to the GluR4 construct, but lacking N-and C-terminal FLAG and c-Myc tags, and with a shorter linker.…”

“…Protein Expression and Purification-The core GluR2 LBD ("S1S2J") that had been used in crystallographic studies of the domain was expressed bacterially, purified, and refolded as described (11,23). The full-length GluR4 LBD construct that had been used in previous solution scattering experiments ("S1S2-D") was generated using the baculovirus expression system and purified as previously described (24).…”

Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS

“…Most importantly, there is no facile method to confirm that the truncations designed have retained the stable elements of the full-length protein. These approaches are problematic in structural genomics efforts, where high-throughput and low cost are dominating considerations (11).…”

Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domain

“…Bacterial expression of soluble S1S2 has been demonstrated in the periplasm, although also at low yields and without glycosylation [14]. Substantially higher yields have been achieved in bacteria by refolding of inclusion-body material, although only for core constructs, in which S1 and S2 are restricted to the regions with PBP sequence homology [15].Such bacterially expressed protein was used to determine the first crystallographic structure of an S1S2 domain core, in complex with the agonist kainate [16]. It confirmed the PBP homology on a structural level, particularly to the glutaminebinding protein QBP.…”