The non-glycosylated N-terminal domain of human thrombopoietin is a molten globule under native conditions

Human thrombopoietin (hTPO) is a primary hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production. The non-glycosylated form of 1–163 residues of hTPO (hTPO₁₆₃) including the N-terminal active site domain (1–153 residues) is a candidate for treating thrombocytopenia. However, the autoantigenicity level of hTPO₁₆₃ is higher than that of the full-length glycosylated hTPO (ghTPO₃₃₂). In order to clarify the structural and physicochemical properties of hTPO₁₆₃, circular dichroism (CD) and differential scanning calorimetry (DSC) analyses were performed. CD analysis indicated that hTPO₁₆₃ undergoes an induced-fit conformational change (+19.0% for helix and −16.7% for β-strand) upon binding to the neutralizing antibody TN1 in a manner similar to the coupled folding and binding mechanism. Moreover, DSC analysis showed that the thermal transition process of hTPO₁₆₃ is a multistate transition; hTPO₁₆₃ is thermally stabilized upon receptor (c-Mpl) binding, as indicated with raising the midpoint (T_m) temperature of the transition by at least +9.5 K. The conformational variability and stability of hTPO₁₆₃ indicate that hTPO₁₆₃ exists as a molten globule under native conditions, which may enable the induced-fit conformational change according to the type of ligands (antibodies and receptor). Additionally, CD and computational analyses indicated that the C-terminal domain (154–332 residues) and glycosylation assists the folding of the N-terminal domain. These observations suggest that the antibody affinity and autoantigenicity of hTPO₁₆₃ might be reduced, if the conformational variability of hTPO₁₆₃ is restricted by mutation and/or by the addition of C-terminal domain with glycosylation to keep its conformation suitable for the c-Mpl recognition.