STRUCTURAL EVIDENCE FOR AN ANION-DIRECTING TRACK IN THE HEN OVOTRANSFERRIN N-LOBE - IMPLICATIONS FOR TRANSFERRIN SYNERGISTIC ANION-BINDING

The transferrins are a class of iron-binding proteins that require the presence of a synergistic anion for conformation-dependent binding of ferric ions. Bromopyruvate, a known synergistic anion and affinity la bel of ovotransferrin (oTF) [Bailey, C. T., Patch, M. G., & Carrano, C . J. (1988) Biochemistry 27, 6276-6282], was used to probe the structu re of the metal- and anion-binding sites of the functional N- and C-te rminal proteolytic halves (oTF/2N and oTF/2C, respectively) of ovotran sferrin. Incubation of oTF/2N with [2-C-14]bromopyruvate in the presen ce of Fe3+ ions resulted in the incorporation of 0.70 mel of C-14 labe l/mol of oTF/2N; C-14-labeled oTF/2N was then purified and digested se quentially with trypsin and V8 protease to determine the sites of modi fication. Quantification of C-14 radioactivity, analysis of purified C -14-labeled peptides by gas-phase sequencing and mass spectrometry dem onstrated that chemical modification was restricted to nucleophilic re sidues contained in a fragment corresponding to residues 189-204 of oT F/2N, including Lys 199, Lys 202, and His 196. Lysine 199 was also pro tected from modification with [H-3]CH2O in iron-saturated oTF/2N, sugg esting the involvement of this residue in anion binding by the ape con formation [Anderson, B. F., Baker, H. M., Norris, G. E., Rumball, S. V ., & Baker, E. N. (1990) Nature 344, 784-787]. Lysine 199 is conserved as a basic residue in the N-terminal metal-binding domains of the tra nsferrins but not in the homologous C-terminal metal-binding domains. Identical trials with oTF/2C showed binding, but not modification, wit h bromopyruvate. These data suggest that Lys 199, Lys 202 and His 196, which are located on an alpha-helix (8) that terminates at the anion- binding site [Dewan, J. C., Mikame, B. Hirose, M., & Sacchettini (1993 ) Biochemistry 32, 11963- 11968], attract and channel the synergistic anion to the anion-binding site. The presence or absence of basic resi dues in the metal-binding lobes of transferrins may account for the di fferent anion- and metal-binding characteristics observed for the iron -binding sites of these proteins.