An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody

Shigeki Arai; Chie Shibazaki; Motoyasu Adachi; Eijiro Honjo; Taro Tamada; Yoshitake Maeda; Tomoyuki Tahara; Takashi Kato; Hiroshi Miyazaki; Michael Blaber; Ryota Kuroki

doi:10.1002/pro.2985

An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody

Shigeki Arai^*, Chie Shibazaki, Motoyasu Adachi, Eijiro Honjo, Taro Tamada, Yoshitake Maeda, Tomoyuki Tahara, Takashi Kato, Hiroshi Miyazaki, Michael Blaber, Ryota Kuroki

^*Corresponding author for this work

School of Education

Research output: Contribution to journal › Article › peer-review

2 Citations (Scopus)

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	https://doi.org/10.1002/pro.2985
Publication status	Published - 2016 Oct 1

Keywords

TN1
X-ray crystallography
antigen–antibody interaction
isothermal titration calorimetry
thrombopoietin

ASJC Scopus subject areas

Biochemistry
Molecular Biology

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10.1002/pro.2985

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@article{f0118c3bbae847f19c2f2f53530599d9,

title = "An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody",

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 {\AA} 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.",

keywords = "TN1, X-ray crystallography, antigen–antibody interaction, isothermal titration calorimetry, thrombopoietin",

author = "Shigeki Arai and Chie Shibazaki and Motoyasu Adachi and Eijiro Honjo and Taro Tamada and Yoshitake Maeda and Tomoyuki Tahara and Takashi Kato and Hiroshi Miyazaki and Michael Blaber and Ryota Kuroki",

note = "Funding Information: Acknowledgments The research of MC has been support by 200930I053 MICINN-CSIC Grant and by CGL2012-37222 Grant from MINECO. PGdS work has funded by grant CGL2007-66440-C04-01 from MICINN. Publisher Copyright: {\textcopyright} 2016 The Protein Society",

year = "2016",

month = oct,

day = "1",

doi = "10.1002/pro.2985",

language = "English",

pages = "1786--1796",

journal = "Protein Science",

issn = "0961-8368",

publisher = "Cold Spring Harbor Laboratory Press",

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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

N1 - Funding Information: Acknowledgments The research of MC has been support by 200930I053 MICINN-CSIC Grant and by CGL2012-37222 Grant from MINECO. PGdS work has funded by grant CGL2007-66440-C04-01 from MICINN. Publisher Copyright: © 2016 The Protein Society

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 - TN1

KW - X-ray crystallography

KW - antigen–antibody interaction

KW - isothermal titration calorimetry

KW - thrombopoietin

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U2 - 10.1002/pro.2985

DO - 10.1002/pro.2985

M3 - Article

C2 - 27419667

AN - SCOPUS:84988432340

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EP - 1796

JO - Protein Science

JF - Protein Science

SN - 0961-8368

ER -

An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody

Abstract

Keywords

ASJC Scopus subject areas

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