Inhibitor screening

The inhibitor screening experiments were initially carried out on the zinc transporting P_IB-2-type ATPase ZntA from Shigella sonnei (SsZntA). SsZntA was produced and purified as described previously (Wang et al., 2014) and the inhibitory effect of approximately 20,000 compounds was assessed by the Chemical biology Consortium Sweden (CBCS). Briefly, the ATPase activity of 0.7 µM highly pure protein was measured in the presence of 50 µM inhibitor through the release of inorganic phosphate (P_i) by the Baginski assay (Baginski et al., 1967) in a total volume of 200 nl as reported earlier (Wang et al., 2014). The inorganic phosphate was detected with Malachite Green reagent (0.005% Carbinol hydrochloride, 1.7% sulfuric acid, 0.14% ammonium molybdate, 0.025% Triton-X) at an absorbance of 620 nm.