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

doi:10.1073/pnas.0308530100

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

^*Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan; and ^‡Pharmaceutical Research Laboratories, Kirin Brewery Co. Ltd., 3 Miyahara-cho, Takasaki 370-1295, Japan

Communicated by Brian W. Matthews, University of Oregon, Eugene, OR, December 20, 2003 (received for review November 12, 2003)

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 × 10⁶ 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.

Footnotes

↵ ¶ To whom correspondence should be addressed. E-mail: r-kuroki{at}kirin.co.jp.
↵ † Present address: deCODE Genetics, Bainbridge Island, WA 98110.
↵ § Present address: Department of Biology, School of Education and Graduate School of Science and Engineering, Waseda University, 1-6-1 Nishi-Waseda, Shinjuku, Tokyo 169-8050, Japan.
Abbreviations: TPO, thrombopoietin; CRH, cytokine receptor homology; hTPO, human TPO; EPO, erythropoietin; hGH, human growth hormone; EPOR, EPO receptor; hGHR, hGH receptor; NCS, noncrystallographic symmetry.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 1V7M and 1V7N).