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

Guo Qiang Chen, Y. U. Sun, Rongsheng Jin, Eric Gouaux

Research output: Contribution to journalArticle

77 Citations (Scopus)

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 HS1S2 (Chen GQ, Gouaux E. 1997. Proc Natl Acad Sci USA 94:13431- 13436) in the presence and absence of glutamate showed that the ligand stabilized the S1 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 S1S2I construct proved to be trypsin- and chymotrypsin-resistant, stable to storage at 4°C, and amenable to three- dimensional crystal 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 Å 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.

Original languageEnglish (US)
Pages (from-to)2623-2630
Number of pages8
JournalProtein Science
Volume7
Issue number12
StatePublished - 1998
Externally publishedYes

Fingerprint

Proteolysis
Mutagenesis
Ligands
Glutamate Receptors
Trypsin
Digestion
Ligand-Gated Ion Channels
Ionotropic Glutamate Receptors
Crystals
alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
Sequence Alignment
Protein Sequence Analysis
Mass spectrometry
Glutamic Acid
Mass Spectrometry
AMPA 2 glutamate receptor ionotropic
Amino Acids
Experiments

Keywords

  • Crystallization
  • Glutamate receptor
  • Ligand binding domain
  • Mutagenesis
  • Protease accessibility

ASJC Scopus subject areas

  • Biochemistry

Cite this

Probing the ligand binding domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct. / Chen, Guo Qiang; Sun, Y. U.; Jin, Rongsheng; Gouaux, Eric.

In: Protein Science, Vol. 7, No. 12, 1998, p. 2623-2630.

Research output: Contribution to journalArticle

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