Abstract
Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody–hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 Å using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (ΔCp) measured by ITC (−1.52 ± 0.05 kJ mol−1 K−1) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (−1.02 ∼ 0.25 kJ mol−1 K−1) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.
| Original language | English |
|---|---|
| Pages (from-to) | 1786-1796 |
| Number of pages | 11 |
| Journal | Protein Science |
| DOIs | |
| Publication status | Published - 2016 Oct 1 |
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Keywords
- antigen–antibody interaction
- isothermal titration calorimetry
- thrombopoietin
- TN1
- X-ray crystallography
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
Cite this
An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody. / Arai, Shigeki; Shibazaki, Chie; Adachi, Motoyasu; Honjo, Eijiro; Tamada, Taro; Maeda, Yoshitake; Tahara, Tomoyuki; Kato, Takashi; Miyazaki, Hiroshi; Blaber, Michael; Kuroki, Ryota.
In: Protein Science, 01.10.2016, p. 1786-1796.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody
AU - Arai, Shigeki
AU - Shibazaki, Chie
AU - Adachi, Motoyasu
AU - Honjo, Eijiro
AU - Tamada, Taro
AU - Maeda, Yoshitake
AU - Tahara, Tomoyuki
AU - Kato, Takashi
AU - Miyazaki, Hiroshi
AU - Blaber, Michael
AU - Kuroki, Ryota
PY - 2016/10/1
Y1 - 2016/10/1
N2 - Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody–hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 Å using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (ΔCp) measured by ITC (−1.52 ± 0.05 kJ mol−1 K−1) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (−1.02 ∼ 0.25 kJ mol−1 K−1) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.
AB - Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody–hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 Å using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (ΔCp) measured by ITC (−1.52 ± 0.05 kJ mol−1 K−1) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (−1.02 ∼ 0.25 kJ mol−1 K−1) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.
KW - antigen–antibody interaction
KW - isothermal titration calorimetry
KW - thrombopoietin
KW - TN1
KW - X-ray crystallography
UR - http://www.scopus.com/inward/record.url?scp=84988432340&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84988432340&partnerID=8YFLogxK
U2 - 10.1002/pro.2985
DO - 10.1002/pro.2985
M3 - Article
SP - 1786
EP - 1796
JO - Protein Science
JF - Protein Science
SN - 0961-8368
ER -