Fig. 7

Putative substrate-binding site located in the C-terminal domain. a Surface representation of the C-terminal domain of BbFpn, viewed from the central cavity. The pocket formed by the non-continuous TM7 is in the center of the domain, with the Ni-EDTA molecule shown in ball-and-stick. Insets show Fo–Fc omit (top) and 2Fo–Fc map (bottom) of the Ni-EDTA site. b Close-up view of the Ni-EDTA-binding site and coordination. Main hydrogen bonds are indicated by dotted lines. R348 forms a salt-bridge to one of the carboxylate groups of the EDTA moiety, shown in yellow, whereas H261 is a direct ligand to the metal (green sphere). c Superposition of the Ni-EDTA bound structure and the previously determined open inward conformation (gray; PDB entry 5AYO) illustrating conformational change of H261. d Effects of mutating residues within the putative metal-binding site of human Fpn. ⁵⁵Fe efflux (black) and ⁶³Ni efflux (green) from control oocytes and oocytes expressing wtFpn or one of several amino-acid substitutions at residue D325 (n = 24, 29, 32, 26, 30, 22, 28, 35, 33, 35, left to right). The bar graph indicates mean, SD, and individual scores. Two-way ANOVA revealed an interaction (P < 0.001). Within ⁵⁵Fe efflux, each mutant differed from wtFpn (P < 0.001); and D325N (P < 0.001) but neither D325A (P = 0.78) nor D325H (P = 0.025) differed from control. Within ⁶³Ni, all groups differed from control (P < 0.001); D325A and D325H differed from wtFpn (P < 0.001) but D325N did not differ from wtFpn (P = 0.14). e–g Working model to illustrate Ca²⁺-activated Fpn-mediated iron efflux