Thermodynamic Analysis of the Activation Mechanism of the GCSF Receptor Induced by Ligand Binding

Shouhei Mine; Takumi Koshiba; Eijiro Honjo; Tomoyuki Okamoto; Taro Tamada; Yoshitake Maeda; Yasuko Matsukura; Akane Horie; Matsujiro Ishibashi; Miharu Sato; Mizue Azuma; Masao Tokunaga; Katsutoshi Nitta; Ryota Kuroki

doi:10.1021/bi0356855

Thermodynamic Analysis of the Activation Mechanism of the GCSF Receptor Induced by Ligand Binding

Shouhei Mine, Takumi Koshiba, Eijiro Honjo, Tomoyuki Okamoto, Taro Tamada, Yoshitake Maeda, Yasuko Matsukura, Akane Horie, Matsujiro Ishibashi, Miharu Sato, Mizue Azuma, Masao Tokunaga, Katsutoshi Nitta, Ryota Kuroki

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Abstract

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 (∼55 Å). 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 A2)] 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 Å) between two CRH domains in the 2:2 complex is much shorter than in our previous model (∼90 Å) predicted from the same crystal structure of the GCSF-CRH complex.

Original language	English
Pages (from-to)	2458-2464
Number of pages	7
Journal	Biochemistry
Volume	43
Issue number	9
DOIs	https://doi.org/10.1021/bi0356855
Publication status	Published - Mar 9 2004
Externally published	Yes

All Science Journal Classification (ASJC) codes

Biochemistry

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10.1021/bi0356855

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@article{34739c3c983d46b680b70b095b32059e,

title = "Thermodynamic Analysis of the Activation Mechanism of the GCSF Receptor Induced by Ligand Binding",

abstract = "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 (∼55 {\AA}). 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 A2)] 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 {\AA}) between two CRH domains in the 2:2 complex is much shorter than in our previous model (∼90 {\AA}) predicted from the same crystal structure of the GCSF-CRH complex.",

author = "Shouhei Mine and Takumi Koshiba and Eijiro Honjo and Tomoyuki Okamoto and Taro Tamada and Yoshitake Maeda and Yasuko Matsukura and Akane Horie and Matsujiro Ishibashi and Miharu Sato and Mizue Azuma and Masao Tokunaga and Katsutoshi Nitta and Ryota Kuroki",

year = "2004",

month = mar,

day = "9",

doi = "10.1021/bi0356855",

language = "English",

volume = "43",

pages = "2458--2464",

journal = "Biochemistry",

issn = "0006-2960",

publisher = "American Chemical Society",

number = "9",

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TY - JOUR

T1 - Thermodynamic Analysis of the Activation Mechanism of the GCSF Receptor Induced by Ligand Binding

AU - Mine, Shouhei

AU - Koshiba, Takumi

AU - Honjo, Eijiro

AU - Okamoto, Tomoyuki

AU - Tamada, Taro

AU - Maeda, Yoshitake

AU - Matsukura, Yasuko

AU - Horie, Akane

AU - Ishibashi, Matsujiro

AU - Sato, Miharu

AU - Azuma, Mizue

AU - Tokunaga, Masao

AU - Nitta, Katsutoshi

AU - Kuroki, Ryota

PY - 2004/3/9

Y1 - 2004/3/9

N2 - 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 (∼55 Å). 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 A2)] 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 Å) between two CRH domains in the 2:2 complex is much shorter than in our previous model (∼90 Å) predicted from the same crystal structure of the GCSF-CRH complex.

AB - 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 (∼55 Å). 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 A2)] 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 Å) between two CRH domains in the 2:2 complex is much shorter than in our previous model (∼90 Å) predicted from the same crystal structure of the GCSF-CRH complex.

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U2 - 10.1021/bi0356855

DO - 10.1021/bi0356855

M3 - Article

C2 - 14992583

AN - SCOPUS:12144286891

SN - 0006-2960

VL - 43

SP - 2458

EP - 2464

JO - Biochemistry

JF - Biochemistry

IS - 9

ER -

Thermodynamic Analysis of the Activation Mechanism of the GCSF Receptor Induced by Ligand Binding

Abstract

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