A Divalent Metal Ion Binding Site in the Kinase Insert Domain of the α-Platelet-Derived Growth Factor Receptor Regulates Its Association with SH2 Domains

To investigate the effects of metal ion binding to the a-PDGFR kinase insert domain, a PCR product representing amino acid residues 691-795 (104 amino acids) was bacterially expressed and purified. Secondary structure prediction and circular dichroism spectroscopy indicated this domain to be a mixed α+β protein with a large coil/tum contribution. This 16 kDa, soluble, nonphosphorylated domain bound to ⁴⁵Ca²⁺ and ⁶⁵Zn²⁺ through a common shared site. Of the unlabeled divalent and trivalent metal ions tested, Ho³⁺ = Zn²⁺ > Ni²⁺ > Ca²⁺ = Mn²⁺ > Mg²⁺, Ba²⁺ in competing for ⁴⁵Ca²⁺ binding to this domain. In the presence of Ca²⁺ ions, the conformation of the KI domain changed significantly, and this changed conformation was resistant to subtilisin proteolysis. However, in the presence of Zn²⁺ ions, the conformation of the KI domain changed only slightly. Nevertheless, Zn²⁺ ions were more effective in rendering the KI domain resistant to proteolysis as compared to that shown by Ca²⁺ ions. In vitro binding studies using purified baculovirus-expressed α-PDGFR showed a marked increase in binding the p85 N-SH2 domain in the presence of Ca²⁺ or Zn²⁺ ions (K_D = 0.5µM), suggesting that metal ion binding enhances association of the p85 N-SH2 domain with the receptor. To confirm this, association of the α-PDGFR with the p85 N-SH2 domain was tested in the presence of the KI domain. The nonphosphorylated KI domain was effective in competing with the α-PDGFR for the binding of the p85 N-SH2 domain. This effect was more pronounced in the presence of Ca²⁺ ions. Microinjection of this domain into Xenopus oocytes delayed maturation in the presence of insulin but not progesterone. This suggests that the KI domain has a correctly folded three-dimensional structure compatible with biological activity. Together these findings indicate that the recombinant α-PDGFR KI domain binds the p85 N-SH2 domain and this binding is modulated by the presence of a novel divalent metal ion binding site within its structure.