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
). Human serum transferrin (hTF) 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 hTF-TFR complex is taken up by receptor-mediated endocytosis culminating in iron release within the endosome (3
). Essential to the reutilization of hTF, iron free hTF (apo-hTF) remains bound to the TFR at low pH. When the apo-hTF-TFR 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
). 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) which is itself stabilized by a conserved arginine residue (6
A key feature of iron binding and release by TF family members is the large conformational change involving not only opening of the two subdomains in each lobe but also a twist between the N1- and N2-, or C1- and C2-subdomains (7
). Although the N- and C-lobes of hTF share 56% sequence similarity, many studies show that the rate of iron release from the C-lobe is considerably slower than the rate of release from the N-lobe, particularly at the putative endosomal pH of ~5.6 (9
). At least some of the difference is attributed to the presence of a “dilysine trigger” in the N-lobe, which is replaced by a triad of residues in the C-lobe (14
). The dilysine trigger is composed of Lys206 in the N2-subdomain and Lys296 in the N1-subdomain which reside on opposite sides of the iron binding cleft and are oriented with side chains extending toward one another allowing them to share a hydrogen bond in the iron bound (closed) conformation (16
). In the C-lobe, a triad of residues (Lys534 in the C2-subdomain, Arg632 and Asp634 in the C1-subdomain) replaces the lysine pair (14
The release of iron from hTF depends upon a number of factors including pH, a chelator (physiologically relevant chelators include citrate, pyrophosphate and ATP), and ionic strength, as well as the specific TFR (19
). Raymond et al
) suggest that a complete model must explain the differences in the rate of iron release from the two lobes, the observed variable chelator concentration dependence, and the effect of anions, as well as, the presence or absence of cooperativity between the lobes.
A 7.5 Å cryo-electron microscopy (cryo-EM) model of diferric hTF bound to TFR was created by docking a human TF N-lobe structure and a rabbit TF C-lobe structure into the electron density map of the complex (since there is no full length human TF structure available) (23
). This model offers a preliminary view of the regions of hTF and TFR which interact; it is suggested that both the N1- and N2- subdomains of the hTF N-lobe contact the TFR, while only the C1-subdomain of the hTF C-lobe appears to be involved in the interaction. Interestingly, a ~9 Å translation of the ferric N-lobe (relative to the ferric C-lobe) is required to dock the two lobes into the cryo-EM density. Of relevance, at pH 7.4, the TFR discriminates between diferric, the two monoferric species, and apo-hTF although the basis of this discrimination has not been explained (24
). Significantly, our studies with authentic monoferric hTF constructs established that each lobe contributes equally (and non-additively) to the binding energy of this interaction with the TFR (15
). Clearly a structure of apo-hTF is required to determine whether a change in orientation of the two lobes could provide both a rationale for discrimination and further insight into the receptor interaction.
Here we report the structure of full-length apo-hTF that has been independently determined by two methods; both a non-glycosylated recombinant form of hTF (pH 6.5) and a glycosylated native form of hTF (pH 7.0) were solved to a resolution of 2.7 Å. These two structures, which are identical within the limits of the resolution, find both the N- and C-lobes in the open conformation. This work represents the first mammalian TF structure with an apo C-lobe, the first published structure of full-length hTF, and the first report of a baby hamster kidney (BHK) expression system to substitute the methionine residues in hTF with selenomethionine (SeMet). The apo-hTF structure allows comparisons to other relevant structures including those for diferric pig (2.15 Å, 72% identical) and rabbit TF (2.6 Å, 79% identical) (27
), and an unpublished model for an unrefined monoferric hTF with iron in the C-lobe (3.3 Å) (28