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.
Highlights
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]
We report the structure of human serum transferrin lacking iron, which was independently determined by two methods: 1) the crystal structure of recombinant non-glycosylated apo-Human serum transferrin (hTF) was solved at 2.7-Aresolution 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 was determined to a resolution of 2.7 Aby molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models
Human serum transferrin4 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
Summary
Production of hTF-NG—To produce recombinant non-glycosylated hTF (hTF-NG) with SeMet substituted for methionine, baby hamster kidney cells transfected with the pNUT plasmid containing the sequence of the N-His-tagged hTF-NG were placed into four expanded surface roller bottles [13]. Adherent baby hamster kidney cells were grown in Dulbecco’s modified Eagle’s medium/ F-12 containing 10% fetal bovine serum. This medium was changed twice at 2-day intervals, followed by addition of Dulbecco’s modified Eagle’s medium/F-12 containing 1% Ultroser G and 1 mM butyric acid. Native apo-hTF-Gly was concentrated in a Centriprep 30 concentrator (Millipore) to 30 mg/ml and screened against commercially available 96-condition screens using a Mosquito crystallization robot (TTP Labtech) with a hanging drop format (drop size, 200 nl of protein plus 200-nl well solution). The crystals used for native data collection were obtained from hanging drops with a well solution of 0.2 M ammonium citrate, pH 7.0, 20% PEG 3350, and 15% glycerol, incubated at 21 °C.
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