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The Transferrin Family
(Sero, Lacto, Ovo & Melano)
Laboratory

Background

Organisms have developed elaborate mechanisms to control the flux of iron. In one of the most important among these mechanisms, the transferrins (TF) sequester Fe(III) avidly (Kd~10-22 M). Thus, Fe(III) is protected from hydrolysis at physiological pH and is unavailable for catalysis of superoxide radical formation via Fenton reactions or for the growth of many pathogens. Serum TFs (STF) bind iron that is absorbed through the gut and transported to various cells. (Note: All actively dividing cells in an organism require iron for the transport of oxygen by hemoglobin, electron transport by cytochromes and the function of ribonucleotide reductase, the rate limiting enzyme in DNA synthesis.) Another family member, lactoferrin (LTF), is found in milk and other bodily secretions. Due to the high affinity with which LTFs bind iron, they are believed to deprive potential pathogens of iron which they require for growth and proliferation. Ovotransferrins (oTF), found in egg whites, may serve the same function, although chicken serum transferrin which shares the same amino acid sequence as OTF also is involved in iron transport. Melanotransferrin (MTF) is found on the surface of melanocytes and its function is unknown.

All TFs feature a bilobal structure with each lobe putatively binding a single iron atom. It has been hypothesized that this bilobal structure is the result of gene duplication. All TFs which have been experimentally shown to bind iron feature the same four amino acid ligands, namely two tyrosines, one histidine and an aspartic acid as well as a synergistic anion which is carbonate. Metal binding has an absolute requirement for the anion which contributes two additional oxygen ligands.

To date, the only confirmed receptors for TFs are those found for serum TFs. The receptors bind diferric-TF, cluster with other receptor complexes, and can be endocytosed in a clathrin coated vesicle. Once internalized, the pH is believed to fall to around 5.6 where an unknown chelator probably assists in the removal of the two iron atoms. The vesicle is then returned to the cell surface where the complexes dissociate and the receptors are free to bind more diferric-TF.

The identification of a "dilysine trigger" in the hSTF N-lobe came from the crystal structure of the proteolytically derived oTF N-lobe and was offered as an explanation for the pH dependence observed for iron release in this lobe. Two lysines, lying in opposite domains, are part of the second shell (defined as residues which interact with liganding residues). In the iron loaded human STF N-lobe structure, the e -amino groups of Lys206 and Lys296 are 3.14 Å apart. This pair is stabilized by a low pKa of one of the Lys-residues, which permits formation of a low energy hydrogen bond. When the pH is lowered, protonation causes the two lysines to repulse each other, providing a positive force that opens the cleft and releases iron. In the apo-protein the lysines are separated by 9 Å. Confirmation of the critical role of this dilysine pair in the mechanism of iron release has been provided by mutagenesis studies. In contrast, although bovine LTF has lysines at equivalent positions, its crystal structure shows that they do not share a hydrogen bond. Why-are they too far apart or misorientated? This structural difference may provide a partial explanation for the slower rate of iron release from LTF.

It is important to note that the C-lobe of human STF does not have an equivalent lysine pair. Instead, a triad of C-lobe residues appear to serve as the pH-sensitive release mechanism. Mutation of any of these three residues to alanine drastically slows iron release or prevents iron release altogether from the C-lobe (our unpublished data).

Data

   None provided. Search the databases for STF, LTF, oTF, MTF and any homologs!

Tools

   Biology Workbench (http://workbench.sdsc.edu)

   GeneDoc (for PCs only:http://www.psc.edu/biomed/genedoc/)

   SwissPDB Viewer (http://us.expasy.org/spdbv/)

   PyMOL (http://pymol.sourceforge.net/)

   NCBI (http://www.ncbi.nlm.nih.gov/)

   PDB (http://www.rcsb.org)

   

Potential Investigations

    • Did a gene duplication occur that forms the N- & C-lobes? If so, when?
    • When did the STF, oTF, MTF & LTF paralogs diverge and become specialized and why?
    • Can you identify other potential members of the transferrin family?
    • What distinguishes an STF, oTF or LTF from one another?
    • What are the physiological functions of oTF & LTF?
    • When did the transferrin receptor arrive on the scene?
    • Can you identify other proteins which use a dilysine trigger as a pH sensitive mechanism to bind and/or release something?

Goals

    • Identify definable questions that can be approached using phylogenetics
    • Find the data & use the tools provided to explore these questions
    • Share your experiments and insights with the group
    • Design and execute experiments to approach these larger questions

 

 
Authors: Stephen J. Everse, John R. Jungck -- 2006