The Catalytic Role Of The M2 Metal Ion In PP2Cα

Protein phosphorylation caused by kinase and protein dephosphorylation caused by phosphatases widely exist in the fundamental life activities, for example, in eukaryotic signaling pathways. What’s more, they commonly regulate the level of protein phosphorylation in cellular. According to the sequence comparison, the sensitivity of the inhibitor and the dependence on metal ion, serine/threonine phosphatases are classified into unrelated PPP (phosphoprotein phosphatases) and PPM super families (metal-dependent protein phosphatases). PP2C family phosphatases (the type 2C family of protein phosphatases; or metal-dependent phosphatase, PPM) is a kind of serine/threonine protein phosphatase and its catalytic activity depends on the metal ions, such as Mg2+or Mn2+. All PP2C family members have a conserved binuclear metal ion active center that is essential for their catalysis. However, the catalytic role of each metal ion during catalysis remains elusive. The PP2Ca is the prototype PP2C family phosphatase for which the catalytic mechanisms and cellular functions have been well characterized. Therefore, we chose PP2Ca as a model for investigating the catalytic role of the M2 metal ion.In our study, we used the D38A and D38K mutations of PP2Ca as specific research tools. First, we constructed flag-PP2Ca with wild-type and mutant plasmids, overexpressed HEK293 and U251 cells and verified cellular functions by western blot. Secondly, we purified wild-type and mutant proteins, and performed crystallizations and analysis of enzyme activity, which includes phosphatase activity for a series of substrates, Ki, pH profile and leaving group dependence.In this study, we discovered that the D38 mutations impair PP2Ca-regulated signaling pathways in cells, and the D38 mutants of PP2Ca decreased the enzyme activity for pNPP, phospho-peptide and phospho-protein substrates. What’s more, the crystal structures of PP2Ca-D38 mutants revealed that the D38A or D38K mutants of PP2Ca redefined the water-mediated hydrogen network in the active site and selectively disrupted M2 metal ion binding. Finally, in combination with enzymology analysis, our results demonstrated that the M2 metal ion participates in dianion substrate binding, determines the rate-limiting step of substrate hydrolysis, and stabilizes the leaving group after P-O bond cleavage. The newly characterized catalytic role of the M2 metal ion in this family not only provides insight into how the binuclear metal centers of the PP2C phosphatases are organized for efficient catalysis but also helps increase our understanding of the function and substrate specificity of PP2C family members.