Ligand binding domain of granulocyte colony-stimulating factor receptor.
The amino-terminal domain of the cytokine receptor homologous region (BN domain; roughly 100 amino acid residues) in the receptor for murine granulocyte colony-stimulating factor (G-CSF) was secreted as a maltose-binding protein fusion into the Escherichia coli periplasm. The murine BN domain (mBN) was prepared from the fusion protein by restriction protease Factor Xa digestion and purified to homogeneity. The purified BN domain specifically and stoichiometrically bound G-CSF, with an apparent dissociation constant (Kd) of 3-8 x 10(-8) M. The CD spectrum of the mBN domain was similar to that of the extracellular region of the human growth hormone (GH) receptor, which is composed of turns and beta-sheets held together by disulfide bonds. Tertiary folding and the beta-sheet of this small domain was confirmed by NMR spectroscopy. Disulfide bonds determined by peptide mapping were in the following locations: Cys107-Cys118, Cys153-Cys162, and Cys143-Cys194. Among them, the first and the second produce small loops (roughly 10 amino acid residues) as found in the human GH receptor. These results suggested that the mBN domain of the G-CSF receptor expressed by E. coli has a GH receptor-like structure. However, the third disulfide bond varied considerably between the G-CSF and GH receptors. Disruption of these disulfide bonds in the BN domain of the G-CSF receptor suggested that all of them are critical for maintaining a stably folded protein. Our results will facilitate understanding of the biophysical and structural properties of this receptor.
Hiraoka
O
O
Protein Engineering Research Institute, Osaka, Japan.
Anaguchi
H
H
Yamasaki
K
K
Fukunaga
R
R
Nagata
S
S
Ota
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D017931:DNA Primers; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D017510:Protein Folding; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins
D017931:DNA Primers; D004220:Disulfides; D008024:Ligands; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D003545:Cysteine