Applied Life Science, Graduate School of Agriculture, Kyoto University
○Bunzo Mikami Kimihiko Mizutani Aiko Tanabe Masaaki Hirose

Ovotransferrin, derived from the same gene as chicken serum transferrin, belongs to transferrin family of iron-binding proteins. They are 80 kDa single-chain bilobe proteins possessing one Fe³⁺ binding site in each lobe. N- and C- lobes have similar tertiary structures and can be isolated after limited proteolysis. Transferrin transports iron ions in blood, and is imported into the cell as complex with transferrin-receptor, and release iron in the acidic endosome through the domain opening initiated by di-lysine trigger. In order to understand the mechanism of iron release, we have refined the structure of ovotransferrin N-lobe at atomic resolution.
For crystallization, ovotransferrin N-lobe was incubated with Fe³⁺ and Na-bicarbonate and concentrated to 18.8 mg/ml. Aliquots were mixed with an equal volume of reservoir solution containing 100 mM Na-HEPES, pH 7.5, 200 mM Na-Acetate, 21-23% PEG3350, and crystallized by hanging-drop vapor diffusion. Red-colored rectangular crystals were obtained. X-ray diffraction data to 0.88 Å resolution were measured at BL38B1 with an imaging plate detector of RIGAKU R-Axis V in SPring-8. Space group of the crystal was P2₁2₁2₁ with lattice parameters: a = 46.144, b = 75.179, c = 84.591 Å. Molecular replacement calculation was succeeded using the structure of holo ovotransferrin N-lobe at low resolution (PDB 1IEJ). Molecular modeling and refinement calculations were performed by Coot and SHELXL. R_cryst/R_free of anisotropic models without hydrogen and with (riding) hydrogen were 11.32%/13.83% and 10.15%/12.18%, respectively, for reflections between infinity - 0.88 Å. Many hydrogen atoms were observed in hydrogen omit map, and the roles of hydrogen atoms in iron-binding and release were investigated.