STRUCTURAL EVIDENCE FOR A PH-SENSITIVE DILYSINE TRIGGER IN THE HEN OVOTRANSFERRIN N-LOBE - IMPLICATIONS FOR TRANSFERRIN IRON RELEASE

Members of the transferrin family of proteins are involved in Fe3+ transport (serum transferrins) and are also believed to possess antimicrobial activity (ovotransferrins and lactoferrins). The structure of the monoferric N-terminal half-molecule of hen ovotransferrin, reported here at 2.3-angstrom resolution, reveals an unusual interdomain interaction formed between the side-chain NZ atoms of Lys 209 and Lys 301, which are 2.3 angstrom apart. This strong interaction appears to be an example of a low-barrier hydrogen bond between the two lysine NZ atoms, both of which are also involved in a hydrogen-bonding interaction with the aromatic ring of a tyrosine residue. Crystals of the protein were grown at pH 5.9, which is well below the usual pK(a) approximately 10 for a lysine side chain. We suggest that the pK(a) of either one or both of these residues lies below the pH of the structure determination and is, therefore, not positively charged. This finding may serve to explain, on a molecular basis, the pH dependence of transferrin Fe3+ release. We propose that uptake of the Fe3+-transferrin complex into an acidic endosome (viz., pH approximately 5.0) via receptor-mediated endocytosis will result in the protonation of both lysine residues. The close proximity of the two resulting positive charges, and their location on opposite domains of the N-lobe, might well be the driving force that opens the two domains of the protein, exposing the Fe3+ ion and facilitating its release. Examination of amino acid sequences of other transferrins indicates that similar pH-sensitive dilysine triggers are possible in the N-lobe, but not in the C-lobe, of most serum transferrins although such interactions have not been reported. Dilysine triggers are not possible in the C-lobe of hen ovotransferrin or in either lobe of most lactoferrins.