Atomic resolution crystal structure of iron-bound ovotransferrin N-lobe

Applied Life Science, Graduate School of Agriculture, Kyoto University
â—‹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. Rcryst/Rfree 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.