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

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 (hTPO(163)) to a 2.5-A resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO(163) 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 hTPO(163) interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 x 10(9) M(-1) and 1.1 x 10(6) M(-1). The presence of the neutralizing Fab did not inhibit binding of hTPO(163) 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.

Fig. 1.

Initial electron density of hTPO₁₆₃ calculated at 2.8 Å and contoured at 1.25 σ. σ_A-Weighted electron density is calculated after solvent flattening, histogram matching, and NCS averaging. The Cα trace for residues 35–123 of the final refined model is overlaid only as a guide.

Fig. 2.

Determinants of the structural frame of hTPO₁₆₃. Stereoviews of the secondary structure show the up-up-down-down topology. Residues that are invariant in the TPO/EPO family are shown in black. Labels indicate the helices of the four-helix bundle (A, B, C, and D) and the minihelix B′. The A/D and B/C helix pairs are shown in blue and green.

Fig. 3.

hTPO₁₆₃–TN1-Fab complex. The Cα traces for the heavy and light chains of the variable domains of the TN1-Fab are shown in green and blue. Only residues that interact with hTPO₁₆₃ are shown. hTPO₁₆₃ is shown in the Corey–Pauling–Koltun molecular model (CPK) with residues that interact with the TN1-Fab highlighted as individually colored CPK atoms. Regions of hTPO₁₆₃ that do not interact with the TN1-Fab are shown as solid orange CPK.

Fig. 4.

Sequence alignment of TPO and EPO from various species. EPO-EPOR site 1 (▴) and site 2 (□) contacts are indicated as described by Syed et al. (22), except for R10 (▪), which contacts both EPOR monomers in the complex. Mutagenic data for TPO (–45) and EPO (, –56) are summarized in a general way with important (○) and critical (•) residues indicated. Secondary structures for TPO and EPO (41) are also indicated for reference.

Fig. 5.

Determinants of specificity of TPO for c-Mpl. Residues that are invariant within the TPO family but not conserved in the EPO family are proposed to confer specificity to the TPO–c-Mpl interaction. These residues fall into two distinct clusters corresponding to the proposed high-affinity (blue) and low-affinity (green) receptor interaction sites. Residues that are invariant in the TPO/EPO family are shown in black.