Thermodynamic analysis of the activation mechanism of the GCSF receptor induced by ligand binding.
The granulocyte colony-stimulating factor receptor (GCSFR), containing the Ig-like domain (Ig) and cytokine receptor homologous region (CRH), was prepared as a preformed dimer (Ig-CRH-Fc)(2) after fusion to the mouse Fc region via an eight-residue linker (approximately 55 A). Monomer Ig-CRH was also prepared after the Fc region was removed from (Ig-CRH-Fc)(2). GCSF binding to Ig-CRH and (Ig-CRH-Fc)(2) was investigated using light scattering and isothermal titration calorimetry. The average molecular mass determined by light scattering showed that both Ig-CRH and (Ig-CRH-Fc)(2) formed a 2:2 dimer with GCSF. Moreover, isothermal titration calorimetry showed that the thermodynamic parameters upon binding of GCSF to Ig-CRH and (Ig-CRH-Fc)(2) were comparable, suggesting a similar binding stoichiometry and interface [including similar buried surface area (5700-6000 A(2))] despite the presence of the eight-residue linker. The buried surface area is much larger than that calculated from our previous report of the crystal structure of the GCSF-CRH complex [Aritomi, M., et al. (1999) Nature 401, 713-717], suggesting a substantial contribution of the Ig domain to GCSF binding. The data also indicate that the distance (55 A) between two CRH domains in the 2:2 complex is much shorter than in our previous model (approximately 90 A) predicted from the same crystal structure of the GCSF-CRH complex.
Pharmaceutical Research Laboratories, Kirin Brewery Company Ltd., 3 Miyahara-cho, Takasaki 370-1295, Japan.
Shouhei
S
Mine
Takumi
T
Koshiba
Eijiro
E
Honjo
Tomoyuki
T
Okamoto
Taro
T
Tamada
Yoshitake
Y
Maeda
Yasuko
Y
Matsukura
Akane
A
Horie
Matsujiro
M
Ishibashi
Miharu
M
Sato
Mizue
M
Azuma
Masao
M
Tokunaga
Katsutoshi
K
Nitta
Ryota
R
Kuroki
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002151:Calorimetry; D002850:Chromatography, Gel; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D008024:Ligands; D008027:Light; D051379:Mice; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D018121:Receptors, Cytokine / Q000235:genetics; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011994:Recombinant Proteins; D012542:Scattering, Radiation; D012996:Solutions; D018411:Spodoptera / Q000235:genetics; D040681:Structural Homology, Protein; D013816:Thermodynamics
D007141:Immunoglobulin Fc Fragments; D008024:Ligands; D018121:Receptors, Cytokine; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012996:Solutions; D016179:Granulocyte Colony-Stimulating Factor