文摘
Human transferrin (Tf) is responsible for the binding and transport of iron in the bloodstreamof vertebrates. Delivery of this bound iron to cells occurs by a process of receptor-mediated endocytosisduring which Tf releases its iron at the reduced endosomal pH of ~5.6. Iron release from Tf involves alarge conformational change in which the two domains that enclose the binding site in each lobe moveapart. We have examined the role of two lysines, Lys206 and Lys296, that form a hydrogen-bonded pairclose to the N-lobe binding site of human Tf and have been proposed to form a pH-sensitive trigger foriron release. We report high-resolution crystal structures for the K206A and K296A mutants of the N-lobehalf-molecule of Tf, hTf/2N, and quantitative iron release data on these mutants and the double mutantK206A/K296A. The refined crystal structures (for K206A, R = 19.6% and Rfree = 23.7%; for K296A, R= 21.2% and Rfree = 29.5%) reveal a highly conserved hydrogen bonding network in the dilysine pairregion that appears to be maintained even when individual hydrogen bonding groups change. The ironrelease data show that the mutants retain iron to a pH 1 unit lower than the pH limit of wild type hTf/2N,and release iron much more slowly as a result of the loss of the dilysine interaction. Added chloride ionsare shown to accelerate iron release close to the pH at which iron is naturally lost and the closed structurebecomes destabilized, and to retard it at higher pH.
