Structure of the Receptor-Binding Domain of Human Thrombopoietin Determined by Complexation with a Neutralizing Antibody Fragment

• Published in: Proceedings of the National Academy of Sciences - PNAS 2004-02, Vol.101 (7), p.1816-1821
• Main Authors: Feese, Michael D., Tamada, Taro, Kato, Yoichi, Maeda, Yoshitake, Hirose, Masako, Matsukura, Yasuko, Shigematsu, Hideki, Muto, Takanori, Matsumoto, Atsushi, Watarai, Hiroshi, Ogami, Kinya, Tahara, Tomoyuki, Kato, Takashi, Miyazaki, Hiroshi, Kuroki, Ryota, Matthews, Brain W.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biochemistry; Biological Sciences; Cells; Coordinate systems; Crossovers; Crystal structure; Crystallography, X-Ray; Crystals; Cytokines; Electron density; Epics; Epitopes - chemistry; Epitopes - immunology; Genetics; Humans; Immunoglobulin Fab Fragments - immunology; Immunoglobulin Fab Fragments - metabolism; Ligands; Models, Molecular; Molecular Sequence Data; Neoplasm Proteins - metabolism; Neutralization Tests; Protein Binding; Protein Conformation; Proto-Oncogene Proteins - metabolism; Receptors; Receptors, Cytokine - metabolism; Receptors, Thrombopoietin; Solubility; Stoichiometry; Structure-Activity Relationship; Substrate Specificity; Thrombopoietin - chemistry; Thrombopoietin - immunology; Thrombopoietin - metabolism; Titration
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0308530100

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 hTPO163has 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 hTPO163interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3× 109M-1and 1.1× 106M-1. The presence of the neutralizing Fab did not inhibit binding of hTPO163to 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.