The PPM phosphatases require millimolar concentrations of Mg2+ or Mn2+ to activate phosphatase activity in vitro. The human phosphatases PP2Cα (PPM1A) and Wip1 (PPM1D) differ in their physiological function, substrate specificity, and apparent metal affinity. A crystallographic structure of PP2Cα shows only two metal ions in the active site. However, recent structural studies of several bacterial PP2C phosphatases have indicated three metal ions in the active site. Two residues that coordinate the third metal ion are highly conserved, suggesting that human PP2C phosphatases may also bind a third ion. Here, isothermal titration calorimetry analysis of Mg2+ binding to PP2Cα distinguished binding of two ions to high affinity sites from the binding of a third ion with a millimolar affinity, similar to the apparent metal affinity required for catalytic activity. Mutational analysis indicated that Asp239 and either Asp146 or Asp243 was required for low-affinity binding of Mg2+, but that both Asp146 and Asp239 were required for catalysis. Phosphatase activity assays in the presence of MgCl2, MnCl2, or mixtures of the two, demonstrate high phosphatase activity toward a phosphopeptide substrate when Mg2+ was bound to the low-affinity site, whether Mg2+ or Mn2+ ions were bound to the high affinity sites. Mutation of the corresponding putative third metal ion-coordinating residues of Wip1 affected catalytic activity similarly both in vitro and in human cells. These results suggest that phosphatase activity toward phosphopeptide substrates by PP2Cα and Wip1 requires the binding of a Mg2+ ion to the low-affinity site.

The p53 tumor suppressor is a critical mediator of the cellular response to stress. The N-terminal transactivation domain of p53 makes protein interactions that promote its function as a transcription factor. Among those cofactors is the histone acetyltransferase p300, which both stabilizes p53 and promotes local chromatin unwinding. Here, we report the nuclear magnetic resonance solution structure of the Taz2 domain of p300 bound to the second transactivation subdomain of p53. In the complex, p53 forms an α-helix between residues 47 and 55 that interacts with the α1-α2-α3 face of Taz2. Mutational analysis indicated several residues in both p53 and Taz2 that are critical for stabilizing the interaction. Finally, further characterization of the complex by isothermal titration calorimetry revealed that complex formation is pH-dependent and releases a bound chloride ion. This study highlights differences in the structures of complexes formed by the two transactivation subdomains of p53 that may be broadly observed and play critical roles in p53 transcriptional activity.

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg 2+ or Mn 2+ ions for activity in vitro. The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn 2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional, third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1A cat , complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1A cat D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1A cat toward a phosphopeptide substrate supported a randomorder, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.Reversible protein phosphorylation signaling pathways are shaped by opposing actions of protein kinases and phosphatases. These pathways regulate the response of cells and organisms to changing environmental and physiological circumstances; dysregulation of these pathways underlies many human diseases. Although phosphorylated protein residues are dominated by phosphoserine (pSer) Trapped PPM1A-phosphopeptide complex 2 of phosphatases involved in reversing the pSer/pThr and pTyr modifications are more evenly divided (1-3). Analysis of global dynamics of pSer/pThr and pTyr-based signaling suggests distinct patterns of regulation (4). The phosphoprotein phosphatases (PPP), with 13 members, and the metal-dependent phosphatases (PPM), with 18 members, provide most of the serine/threonine protein phosphatase activity in human cells (5,6).The activity, substrate specificity, and subcellular localization of PPP phosphatases are regulated primarily by binding of regulatory or inhibitor subunits, of which over 100 have been identified in human cells (7,8). PPM phosphatases generally are monomeric, but regulation of their activity remains incompletely understood (9-11). The evolutionarily distinct PPP and PPM phosphatases both feature tightly-bound bi-metal clusters in their active sites, but only the PPM phosphatases req...