Mn²⁺-binding properties of a recombinant protein-tyrosine kinase derived from the human insulin receptor

The divalent cation-binding properties of the human insulin receptor tyrosine kinase domain were examined kinetically and by electron paramagnetic resonance and circular dichroic spectroscopy. The protein-tyrosine kinase activity of the purified cytoplasmic domain can be activated nearly 10-fold by 3 mM Mn²⁺ in the presence or absence of 5 mM Mg²⁺. Electron paramagnetic resonance spectra of the purified, acid-denatured kinase domain and assays of EDTA-treated kinase show that the purified protein does not possess residual, tightly bound Mn2+. Electron paramagnetic resonance spectroscopy was used to directly measure the binding constant of the kinase domain for Mn²⁺. The results indicate that the recombinant cytoplasmic domain of the human insulin receptor does not bind Mn²⁺ tightly in the absence or presence of MgATP (K_d > 0.8 mM). Furthermore, the enzyme does not show a strong preference for MnATP binding when both MgATP and MnATP are present. The far-ultraviolet circular dichroic spectrum of this domain is characterized by a negative maximum at 207 nm. In the presence of Mn²⁺, but not Mg²⁺, changes in the mean residue-weight ellipticity at 207 nm occur that are consistent with a decrease in α-helical content. The addition of ATP to Mn²⁺-bound protein does not further perturb the spectrum. We conclude that Mn²⁺ ions, although they bind weakly, induce an activating conformational change in the secondary structure of the human insulin receptor cytoplasmic domain. Activation by Mn²⁺ is unlikely to be significant in intact cells, but it may mimic the action of a physiological activator.