Dual role of Lys206-Lys296 interaction in human transferrin N-lobe: Iron-release trigger and anion-binding site

The unique structural feature of the dilysine (Lys206-Lys296) pair in the t ransferrin N-lobe (hTF/2N) has been postulated to serve a special function in the release of iron from the protein. These two lysines, which are locat ed in opposite domains, hydrogen bond to each other in the iron-containing hTF/2N at neutral pH but are far apart in the ape-form of the protein. It h as been proposed that charge repulsion resulting from the protonation of th e dilysines at lower pH may be the trigger to open the cleft and facilitate iron release. The fact that the dilysine pair is positively charged and re sides in a location close to the metal-binding center has also led to the s uggestion that the dilysine pair is an anion-binding site for chelators. Th e present report provides comprehensive evidence to confirm that the dilysi ne pair plays this dual role in modulating release of iron. When either of the lysines is mutated to glutamate or glutamine or when both are mutated t o glutamate, release of iron is much slower compared to the wildtype protei n. This is due to the fact that the driving force for cleft opening is abse nt in the mutants or is converted to a lock-like interaction (in the case o f the K206E and K296E mutants). Direct titration of the ape-proteins with a nions as well as anion-dependent iron release studies show that the dilysin e pair is part of an active anion-binding site which exists with the Lys296 -Tyr188 interaction as a core. At this site, Lys296 serves as the primary a nion-binding residue and Tyr188 is the main reporter for electronic spectra l change, with smaller contributions from Lys206, Tyr85, and Tyr95. In iron -loaded hTF/2N, anion binding becomes invisible as monitored by UV-vis diff erence spectra since the spectral reporters Tyr188 and Tyr95 are bound to i ron. Our data strongly support the hypothesis that the apo-hTF/2N exists in equilibrium between the open and closed conformations, because only in the closed form is Lys296 in direct. contact with Tyr188. The current findings bring together observations, ideas, and experimental data from a large num ber of previous studies and shed further light on the detailed mechanism of iron release from the transferrin N-lobe. In iron-containing hTF/2N, Lys29 6 may still function as a target to introduce an anion (or a chelator) near to the iron-binding center. When the pH is lowered, the protonation of car bonate (synergistic anion for metal binding) and then the dilysine pair for m the driving force to loosen the cleft, exposing iron; the nearby anion (o r chelator) then binds to the iron and releases it from the protein.