Amino acid residues on the surface of soybean 4-kDa peptide involved in the interaction with its binding protein.
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.
Hanada
Kazuki
K
Yokohama City University, Kihara Institute for Biological Research/Graduate School of Integrated Science, Yokohama, Japan.
Nishiuchi
Yuji
Y
Hirano
Hisashi
H
D000418:Albumins; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D017931:DNA Primers; D014644:Genetic Variation; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013025:Glycine max / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance
D000418:Albumins; D002352:Carrier Proteins; D017931:DNA Primers; C089264:PA1 protein, Glycine max; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases