Fig. 4: Amino acid 93 modulates Ca²⁺ binding in the low-affinity Ca²⁺ -binding pocket and stabilizes the NA tetramer under Ca²⁺-depleted conditions.

a NA protein model showing the interaction of G111, G112, and D113 with water molecules in the symmetry axis of the NA tetramer of hVIC/11 (right panel, green) and sOH/04 (left panel, blue). Water molecules are shown in red, and Ca²⁺ is shown in green. b sOH/04 (blue) and sOH/04-D113A (pink) NA sialidase activity at different Ca²⁺ concentrations. Values are presented as a percentage of the reaction velocity compared to 2 mM Ca²⁺ which was set at 100%, measured at 37 °C. Values represent the mean ± SD of three independent experiments. c sOH/04 and sOH/04-D113A thermostability assessed at 2 mM Ca²⁺ (solid line) or 0 mM Ca²⁺ (dashed line). T50 was determined by adjusting the data of three independent experiments to a dose-response variable slope nonlinear fit. d Left panel: Thermostability of hVIC/11 (yellow), hVIC/11-G93N (brown), and hVIC/11-A138S/G93N (green). Right panel: Thermostability of sOH/04 (blue) and sOH/04-N93G (orange). Thermostability profiles were determined at 2 mM Ca²⁺ (solid line) or 0 mM Ca²⁺ (dashed line). Values represent the mean ± SD of three independent experiments. e T50 values were determined by adjusting the data of three independent experiments to a dose-response variable slope nonlinear fit. p values were obtained by ordinary one-way ANOVA. f Left panel: NA sialidase activity of hVIC/11 (yellow), hVIC/11-G93N (brown), and hVIC/11-A138S/G93N (green) at different Ca²⁺ concentrations. Right panel: NA sialidase activity of sOH/04 (blue) and sOH/04-N93G (orange) at different Ca²⁺ concentrations. Values are expressed as the reaction velocity percentage compared to the velocity at 2 mM Ca²⁺ (100% activity), measured at 37 °C. Values represent the mean ± SD of three independent experiments. (g) NA activity at 0 mM Ca²⁺ of hVIC/11 and sOH/04 NA 93 mutants expressed as a percentage of the reaction velocity compared to 2 mM Ca²⁺ (100% NA activity), measured at 37 °C. Values are shown as the means of three independent experiments and p values were obtained by ordinary one-way ANOVA.