The Crystal Structure of Iron-free Human Serum Transferrin Provides Insight into Inter-lobe Communication and Receptor Binding

Abstract

Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependent process. The binding and release of iron result in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF), which was independently determined by two methods: 1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7-Å resolution using a multiple wavelength anomalous dispersion phasing strategy, by substituting the nine methionines in hTF with selenomethionine and 2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7 Å by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human transferrin and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4°and 49.5°rotations are required to open the N-and C-lobes, respectively (compared with closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N-and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor.The transferrins are a family of bilobal iron-binding proteins that play the crucial role of binding ferric iron and keeping it in solution, thereby controlling the levels of this important metal in the body (1, 2). Human serum transferrin (hTF) 4 is synthesized in the liver and secreted into the plasma; it acquires Fe(III) from the gut and delivers it to iron requiring cells by binding to specific transferrin receptors (TFR) on their surface. The entire hTF⅐TFR complex is taken up by receptor-mediated endocytosis culminating in iron release within the endosome (3). Essential to the re-utilization of hTF, iron-free hTF (apo-hTF) remains bound to the TFR at low pH. When the apo-hTF⅐TFR complex is returned to the cell surface, apo-hTF is released to acquire more iron.Strong homologies exist, both between TF family members, and between the two lobes of any given TF (4, 5). Each N-and C-lobe is divided into two subdomains (designated N1 and N2, and C1 and C2) connected by a hinge that gives rise to a deep cleft containing the iron-binding ligands. Iron is coordinated by four highly conserved amino acid residues: an aspartic acid (the sole ligand from the N1-or C1-subdomain), a tyrosine in the hinge at the edge of the N2-or C2-subdomain, a second tyrosine within the N2-or C2-subdomain, and a histidine at the hinge bordering the N1-or C1-subdomain. In addition, the iron atom is bound by two oxygen atoms from the synergistic anion (carbonate), w...

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How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH

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