Enzymes: alkaline phosphatase (EC 184.108.40.206); lysylendopeptidase (EC 220.127.116.11); restriction endonucleases EcoRI (EC 18.104.22.168) and NcoI (EC 22.214.171.124); thioredoxin reductase (EC 126.96.36.199); tyrosine kinase (EC 188.8.131.52).
Amino acid residues on the surface of soybean 4-kDa peptide involved in the interaction with its binding protein
Article first published online: 21 MAY 2003
European Journal of Biochemistry
Volume 270, Issue 12, pages 2583–2592, June 2003
How to Cite
Hanada, K., Nishiuchi, Y. and Hirano, H. (2003), Amino acid residues on the surface of soybean 4-kDa peptide involved in the interaction with its binding protein. European Journal of Biochemistry, 270: 2583–2592. doi: 10.1046/j.1432-1033.2003.03627.x
Note: As the binding capabilities of insulin and the 4-kDa peptide to the 43 kDa protein were similar, Watanabe et al. named the 4-kDa peptide as leginsulin in their early publication. There are many controversies related to the naming of this peptide as leginsulin. To avoid confusion, in the present article we referred to the peptide as ‘4-kDa peptide’ instead of leginsulin.
- Issue published online: 21 MAY 2003
- Article first published online: 21 MAY 2003
- (Received 28 February 2003, revised 16 April 2003, accepted 22 April 2003)
- hormone-like peptide;
- receptor-like protein;
- protein–protein interaction;
- alanine-scanning mutagenesis;
- surface plasmon resonance
Soybean 4-kDa peptide, a hormone-like peptide, is a ligand for the 43-kDa protein in legumes that functions as a protein kinase and controls cell proliferation and differentiation. As this peptide stimulates protein kinase activity, the interaction between the 4-kDa peptide (leginsulin) and the 43-kDa protein is considered important for signal transduction. However, the mechanism of interaction between the 4-kDa peptide and the 43-kDa protein is not clearly understood. We therefore investigated the binding mechanism between the 4-kDa peptide and the 43-kDa protein, by using gel-filtration chromatography and dot-blot immunoanalysis, and found that the 4-kDa peptide bound to the dimer form of the 43-kDa protein. Surface plasmon resonance analysis was then used to explore the interaction between the 4-kDa peptide and the 43-kDa protein. To identify the residues of the 4-kDa peptide involved in the interaction with the 43-kDa protein, alanine-scanning mutagenesis of the 4-kDa peptide was performed. The 4-kDa peptide-expression system in Escherichia coli, which has the ability to install disulfide bonds into the target protein in the cytoplasm, was employed to produce the 4-kDa peptide and its variants. Using mass spectrometry, the expressed peptides were confirmed as the oxidized forms of the native peptide. Surface plasmon resonance analysis showed that the C-terminal hydrophobic area of the 4-kDa peptide plays an important role in binding to the 43-kDa protein.