Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment

Michael D. Feese, Taro Tamada, Yoichi Kato, Yoshitake Maeda, Masako Hirose, Yasuko Matsukura, Hideki Shigematsu, Takanori Muto, Atsushi Matsumoto, Hiroshi Watarai, Kinya Ogami, Tomoyuki Tahara, Takashi Kato, Hiroshi Miyazaki, Ryota Kuroki

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Abstract

The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO163) to a 2.5-Å resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO163 has an antiparallel four-helix bundle fold. The neutralizing Fab mainly recognizes the C-D crossover loop containing the species invariant residue Q111. Titration calorimetric experiments show that hTPO163 interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 × 109 M-1 and 1.1 × 10 6 M-1. The presence of the neutralizing Fab did not inhibit binding of hTPO163 to soluble c-Mpl fragments, but the lower-affinity binding disappeared. Together with prior genetic data, these define the structure-function relationships in TPO and the activation scheme of c-Mpl.

Original languageEnglish
Pages (from-to)1816-1821
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number7
DOIs
Publication statusPublished - 2004 Feb 17

ASJC Scopus subject areas

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    Feese, M. D., Tamada, T., Kato, Y., Maeda, Y., Hirose, M., Matsukura, Y., Shigematsu, H., Muto, T., Matsumoto, A., Watarai, H., Ogami, K., Tahara, T., Kato, T., Miyazaki, H., & Kuroki, R. (2004). Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment. Proceedings of the National Academy of Sciences of the United States of America, 101(7), 1816-1821. https://doi.org/10.1073/pnas.0308530100