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

Daruka Mahadevan, Narmada Thanki, Pilar Aroca, Peter McPhie, Jin Chen Yu, John Beeler, Eugenio Santos, Alexander Wlodawer, Mohammad A. Heidaran

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2095-2106
Number of pages12
JournalBiochemistry
Volume34
Issue number7
DOIs
StatePublished - Feb 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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