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-A 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 2.7A 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 degrees and 49.5 degrees 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.

Fig. 1

Stereo image of the crystal structure of apo-hTF with subdomain N1 in red, N2 in blue, C1 in green and C2 in yellow. The linker region between the lobes is colored grey and the hinge between the N1- and N2- subdomains and the C1- and C2-subdomains is identified. Four of the citrate molecules associated with apo-hTF are shown by stick representation. All figures were made with PYMOL (89).

Fig. 2

Stereo image of the structural superposition of apo-hTF with holo-pig TF created by aligning the C1-subdomains of the two structures and then superimposing the full molecules on their own C1-subdomains. Apo-hTF is blue and holo-pig TF is red. Note that when the C1-subdomains are superimposed, the N1-subdomains fall into register, but the N2- and C2-subdomains are misaligned. The overlay used SPDBV (90)

Fig. 3

A superimposition of the N-lobe and C-lobe of apo-hTF illustrating the difference in secondary structure of the region surrounding the hinge residues between the two subdomains of each lobe. In the N-lobe (N1- blue and N2- red), the hinge is adjacent to an anti-parallel β-sheet formed from β-strands e and j. In the C-lobe (C1- green and C2- yellow) the hinge is located within an unstructured region, as β-strand e is shortened and β-strand j is entirely absent. The overlay used SPDBV (90)

Fig. 4

The interface between the two-lobes of apo-hTF is shown to illustrate the inter-lobe contacts. Two pairs of salt bridged side chains are present at the interface: Arg308 in the N1-subdomain to Asp376 in the C1-subdomain and Asp240 in the N2- subdomain to Arg678 in the C1-subdomain. The subdomains are colored as in Figure 1 and the salt bridging side chains are shown in stick representation. The inset shows the location of the inter-lobe contacts the context of the complete apo-hTF structure.