Figure 4. Putative sodium binding sites in the StOAD β subunit.

(a) Cutaway view of the solvent-accessible surface of the StOAD β₃ sub-complex. The surface is colored according to the electrostatic potential. Helical hairpins H1, H2 and the Asp203 and Ser382 side chains at their tips are highlighted. The γ subunits are presented in cartoon representation and colored in blue. The DDM molecules in the cavity at the center of the complex are presented in stick representation with their carbon atoms in gray. (b) Structure of the putative sodium binding sites in the β subunit. The gray spheres 1 and 2 indicate equivalent positions of the first and second sodium binding sites in CitS, respectively. Polar or charged groups in the proximity are highlighted. (c) ITC experiments probing sodium binding to the wild type StOAD βγ sub-complex and variants with substitutions at the putative β subunit sodium binding sites. Errors are derived from fitting the data to the one set of sites binding model. (d) Oxaloacetate decarboxylation by the wild type StOAD and variants with substitutions at the putative β subunit sodium binding sites. The activity was reconstituted by supplementing the α subunit with detergent-solubilized βγ sub-complex. The specific activity is defined as the reaction velocity divided by the amount of StOAD. In the control experiment (Ctrl), the βγ sub-complex was omitted in the reaction mixture. The error bars represent standard deviations of three independent experiments. C_1/2, sodium concentration at half-maximum activity; V_max, maximum specific activity. Values of C_1/2 and V_max and their errors were derived from data fitting.

Figure 4—figure supplement 1. Thermograms of ITC experiments probing sodium binding to the wild type and substituted StOAD βγ sub-complex.

The Reference experiment (Ref) was performed in the absence of the StOAD βγ sub-complex.