Definition of the residues required for the interaction between glycine-extended gastrin and transferrin in vitro.
ABSTRACT Transferrin is the main iron transport protein found in the circulation, and the level of transferrin saturation in the blood is an important indicator of iron status. The peptides amidated gastrin(17) (Gamide) and glycine-extended gastrin(17) (Ggly) are well known for their roles in controlling acid secretion and as growth factors in the gastrointestinal tract. Several lines of evidence, including the facts that transferrin binds gastrin, that gastrins bind ferric ions, and that the level of expression of gastrins positively correlates with transferrin saturation, suggest the possible involvement of the transferrin-gastrin interaction in iron homeostasis. In the present work, the interaction between gastrins and transferrin has been characterized by surface plasmon resonance and covalent crosslinking. First, an interaction between iron-free apo-transferrin and Gamide or Ggly was observed. The fact that no interaction was observed in the presence of the chelator EDTA suggested that the gastrin-ferric ion complex was the interacting species. Moreover, removal of ferric ions with EDTA reduced the stability of the complex between apo-transferrin and gastrins, and no interaction was observed between Gamide or Ggly and diferric transferrin. Second, some or all of glutamates at positions 8-10 of the Ggly molecule, together with the C-terminal domain, were necessary for the interaction with apo-transferrin. Third, monoferric transferrin mutants incapable of binding iron in either the N-terminal or C-terminal lobe still bound Ggly. These findings are consistent with the hypothesis that gastrin peptides bind to nonligand residues within the open cleft in each lobe of transferrin and are involved in iron loading of transferrin in vivo.
Article: The crystal structure of iron-free human serum transferrin provides insight into inter-lobe communication and receptor binding.[show abstract] [hide abstract]
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.Journal of Biological Chemistry 09/2006; 281(34):24934-44. · 4.77 Impact Factor