Fig. 7: Roles of the His-loop in Zn²⁺ binding affinity and Zn²⁺ uptake activity of hZnT7.

a Cartoon representations show the distribution of histidine residues in the His-loop, and the constructions of HS1, HS2, HS3 and AAAA mutants of hZnT7. b ITC analyses for Zn²⁺ binding to hZnT7 WT and its His-to-Ser mutants (HS1, HS2 and HS3) and AAAA mutant with mutations of the HDHD motif to alanine. The upper panels show the heat generation during the calorimetric titrations with 1 μL injections of 500 μM ZnCl₂ into hZnT7 WT (18 μM), HS1 (20 μM), HS2 (20 μM), HS3 (30 μM) and AAAA (20 μM) in the reaction cell with a volume of 280 μL. The lower panels display the integrated heat values calculated from the upper panels as a function of the molar ratio of Zn²⁺ to hZnT7. The solid lines represent the best fit to the observed ITC curves. At least two independent replicates for each construct. c The resulting fitting parameters are summarized. d Initial Zn²⁺ uptake rates per μg of hZnT7 protein determined with the proteoliposomes containing the indicated ZnT7 protein and Fluozin3 in buffer containing 100 μM Zn²⁺. Data are averages from three independent experiments. Error-bars indicate mean ± SEM (n = 3); numbers indicate p values analyzed by one-way ANOVA. Source numerical data are provided in the Source Data file. See also Supplementary Fig. 21.