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 (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.
Feese
Michael D
MD
Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan.
Tamada
Taro
T
Kato
Yoichi
Y
Maeda
Yoshitake
Y
Hirose
Masako
M
Matsukura
Yasuko
Y
Shigematsu
Hideki
H
Muto
Takanori
T
Matsumoto
Atsushi
A
Watarai
Hiroshi
H
Ogami
Kinya
K
Tahara
Tomoyuki
T
Kato
Takashi
T
Miyazaki
Hiroshi
H
Kuroki
Ryota
R
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D009500:Neutralization Tests; D011485:Protein Binding; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D018121:Receptors, Cytokine / Q000378:metabolism; D053628:Receptors, Thrombopoietin; D012995:Solubility; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013926:Thrombopoietin / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*
D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; D013926:Thrombopoietin