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 label of ovotransferrin (oTF) [Bailey, C. T., Patch, M. G., & Carrano, C. J. (1988) Biochemistry 27, 6276-6282], was used to probe the structure of the metal- and anion-binding sites of the functional N- and C-terminal proteolytic halves (oTF/2N and oTF/2C, respectively) of ovotransferrin. Incubation of oTF/2N with [2-14C]bromopyruvate in the presence of Fe3+ ions resulted in the incorporation of 0.70 mol of 14C label/mol of oTF/2N; 14C-labeled oTF/2N was then purified and digested sequentially with trypsin and V8 protease to determine the sites of modification. Quantification of 14C radioactivity, analysis of purified 14C-labeled peptides by gas-phase sequencing and mass spectrometry demonstrated that chemical modification was restricted to nucleophilic residues contained in a fragment corresponding to residues 189-204 of oTF/2N, including Lys 199, Lys 202, and His 196. Lysine 199 was also protected from modification with [3H]CH2O in iron-saturated oTF/2N, suggesting the involvement of this residue in anion binding by the apo conformation [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 transferrins but not in the homologous C-terminal metal-binding domains. Identical trials with oTF/2C showed binding, but not modification, with 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 residues in the metal-binding lobes of transferrins may account for the different anion- and metal-binding characteristics observed for the iron-binding sites of these proteins.
Department of Biochemistry, University of Vermont, Burlington 05405, USA.
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Nadeau
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Falick
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Woodworth
D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D002645:Chickens; D003207:Conalbumin / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008745:Methylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011773:Pyruvates / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D000838:Anions; D010446:Peptide Fragments; D011773:Pyruvates; D003207:Conalbumin; D014443:Tyrosine; D006639:Histidine; C017092:bromopyruvate; D008239:Lysine