FIG 2.
c-di-AMP binds specifically and with a Kd of 2 ± 0.18 μM to the recombinant S. aureus MBP-KdpD(USP) protein. (A) c-di-AMP binding curve and Kd determination using the purified MBP-KdpD(USP) fusion protein. DRaCALAs were performed with radiolabeled c-di-AMP and protein concentrations ranging from 0.02 to 150 μM. The average fraction-bound values and standard deviations from three independent experiments were plotted against the protein concentration, the binding curve was fitted by using one-site-specific binding nonlinear regression, and the Kd value was determined as previously described (21, 41). (B) DRaCALAs were performed with the purified MBP-KdpD(USP) protein, radiolabeled c-di-AMP, and an excess (100 μM) of the indicated cold competitor nucleotide. Two independent experiments were performed. Representative DRaCALA spots are shown, and the average fraction-bound values and standard deviations from triplicates are plotted.