Alternative structural state of transferrin: The crystallographic analysis of iron-loaded but domain-opened ovotransferrin N-lobe

Transferrins bind Fe³⁺ very tightly in a closed interdomain cleft by the coordination of four protein ligands (Asp⁶⁰, Tyr⁹², Tyr¹⁹¹, and His²⁵⁰ in ovotransferrin N-lobe) and of a synergistic anion, physiologically bidentate CO₃/^2-. Upon Fe³⁺ uptake, transferrins undergo a large scale conformational transition: the apo structure with an opening of the interdomain cleft is transformed into the closed holo structure, implying initial Fe³⁺ binding in the open form. To solve the Fe³⁺-loaded, domain- opened structure, an ovotransferrin N-lobe crystal that had been grown as the apo form was soaked with Fe³⁺-nitrilotriacetate, and its structure was solved at 2.1 Å resolution. The Fe³⁺-soaked form showed almost exactly the same overall open structure as the iron-free apo form. The electron density map unequivocally proved the presence of an iron atom with the coordination by the two protein ligands of Tyr⁹²-OH and Tyr¹⁹¹-OH. Other Fe³⁺ coordination sites are occupied by a nitrilotriacetate anion, which is stabilized through the hydrogen bonds with the peptide NH groups of Ser¹²², Ala¹²³, and Gly¹²⁴ and a side chain group of Thr¹¹⁷. There is, however, no clear interaction between the nitrilotriacetate anion and the synergistic anion binding site, Arg¹²¹.