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 (KD = 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.
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