Abstract
Both PPM phosphatases and their opposing kinases regulate key cellular events in both eukaryotes and prokaryotes. While kinases have a deep active site groove where their substrates dock, this is not the case for phosphatases. The mechanism behind how PPM phosphatases achieve specificity for their substrates is unknown. Bacillus subtilis only has five PPM phosphatases, making it an ideal system to explore how these phosphatases achieve specificity. RsbU and SpoIIE, are paralogous phosphatases that regulate the general stress response and sporulation in Bacillus subtilis, respectively. They dephosphorylate two structurally similar substrates, RsbV and SpoIIAA, with exquisite specificity. It is critical for these two pathways to avoid crosstalk as activation of σF during vegetative growth is toxic to the cells, and activation of B will halt sporulation. By combining the power of genetics, biochemical approaches, and AlphaFold2 structure predictions, we identified three variable loops at the substrate docking interface on the surface of the PPM phosphatase that recognizes the substrate's three-dimensional structure. These three variable loops are conserved elements that contain non-conserved amino acids that either allow accommodation of the cognate substrate or disfavor accommodation of the non-cognate substrate. When the substrate engages these three variable loops in the phosphatase domain, it docks against the switch, positioning the substrate to organize the catalytic center.