Ligand variation in the transferrin family: The crystal structure of the H249Q mutant of the human transferrin N-lobe as a model for iron binding in insect transferrins

Proteins of the transferrin (Tf) family play a central role in iron homeost asis in vertebrates. In vertebrate Tfs, the four iron-binding ligands, 1 As p, 2 Tyr, and I His, are invariant in both lobes of these bilobal proteins. In contrast, there are striking variations in the Tfs that have been chara cterized from insect species; in three of them, sequence changes in the C-l obe binding site render it nonfunctional, and in all of them the His ligand in the N-lobe site is changed to Gln. Surprisingly, mutagenesis of the his tidine ligand, His249, to glutamine in the N-lobe half-molecule of human Tf (hTf/2N) shows that iron binding is destabilized and suggests that Gln249 does not bind to iron. We have determined the crystal structure of the H249 Q mutant of hTf/2N and refined it at 1.85 Angstrom resolution (R = 0.221, R -free = 0.246). The structure reveals that Gln249 does coordinate to iron, albeit with a lengthened Fe-O epsilonl bond of 2.34 Angstrom. In every othe r respect, the protein structure is unchanged from wild-type. Examination o f insect Tf sequences shows that the K206...K296 dilysine pair, which aids iron release from the N-lobes of vertebrate Tfs, is not present in the inse ct proteins. We conclude that substitution of Gln for His does destabilize iron binding, but in the insect Tfs this is compensated by the loss of the dilysine interaction. The combination of a His ligand with the dilysine pai r in vertebrate Tfs may have been a later evolutionary development that giv es more sophisticated pH-mediated control of iron release from the N-lobe o f transferrins.