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

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.