Fig. 6: Proposed mode of recognition Ca²⁺ and inorganic phosphate ion (P_i) by inward-facing YfkE.

a P_i transport kinetic assays measured using inside-out vesicles: N69A, N99A, N252A, Q281A, and H256A. Different concentrations (5× higher than WT) of vesicles were used for mutants to obtain useful data for kinetic analysis. Data were subtracted with (−) control vesicles and then fitted into the Michalis–Menten kinetics using GraphPad 9. Error bars represent standard deviations (n = 3). b Overview of the YfkE model viewed along the membrane plane; the two inverted-topology repeats, each comprising five transmembrane helices, are colored in orange (TM1-TM5) and marine blue (TM6-10). A transmembrane helix (TM0) precedes the N-terminal repeat. The binding sites for Ca²⁺ (magenta sphere) and P_i (yellow and red spheres) are formed by residues in the so-called alpha-repeats, i.e., TM2-TM3 and TM7-TM8. c Close-up of the hypothetical structure of the binding site for Ca²⁺ and P_i in YfkE. Residues involved in ion coordination are highlighted. The proposed interaction network is indicated with black lines. d Same as c, with overlaid occupancy maps calculated from the ensemble of models generated in this study. Specifically, the figure shows a contour of the occupancy maps at a 85% value, for both the protein side-chains (gray mesh), Ca²⁺ (green mesh), and P_i (red mesh). That is, the portion of the structure inside the contours is roughly consistent across 85% of the ensemble, while the portion outside is variable.