| Abstract
| - Proteins of the transferrin (Tf) family play a central role in iron homeostasis in vertebrates. Invertebrate Tfs, the four iron-binding ligands, 1 Asp, 2 Tyr, and 1 His, are invariant in both lobes of thesebilobal proteins. In contrast, there are striking variations in the Tfs that have been characterized frominsect species; in three of them, sequence changes in the C-lobe binding site render it nonfunctional, andin all of them the His ligand in the N-lobe site is changed to Gln. Surprisingly, mutagenesis of the histidineligand, His249, to glutamine in the N-lobe half-molecule of human Tf (hTf/2N) shows that iron bindingis destabilized and suggests that Gln249 does not bind to iron. We have determined the crystal structureof the H249Q mutant of hTf/2N and refined it at 1.85 Å resolution (R = 0.221, Rfree = 0.246). Thestructure reveals that Gln249 does coordinate to iron, albeit with a lengthened Fe−Oε1 bond of 2.34 Å.In every other respect, the protein structure is unchanged from wild-type. Examination of insect Tf sequencesshows that the K206···K296 dilysine pair, which aids iron release from the N-lobes of vertebrate Tfs, isnot present in the insect proteins. We conclude that substitution of Gln for His does destabilize ironbinding, but in the insect Tfs this is compensated by the loss of the dilysine interaction. The combinationof a His ligand with the dilysine pair in vertebrate Tfs may have been a later evolutionary developmentthat gives more sophisticated pH-mediated control of iron release from the N-lobe of transferrins.
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