Allosteric inhibition of PPM1D serine/threonine phosphatase via an altered conformational state.

PPM1D encodes a serine/threonine phosphatase that regulates numerous pathways including the DNA damage response and p53. Activating mutations and amplification of PPM1D are found across numerous cancer types. GSK2830371 is a potent and selective allosteric inhibitor of PPM1D, but its mechanism of binding and inhibition of catalytic activity are unknown. Here we use computational, biochemical and functional genetic studies to elucidate the molecular basis of GSK2830371 activity. These data confirm that GSK2830371 binds an allosteric site of PPM1D with high affinity. By further incorporating data from hydrogen deuterium exchange mass spectrometry and sedimentation velocity analytical ultracentrifugation, we demonstrate that PPM1D exists in an equilibrium between two conformations that are defined by the movement of the flap domain, which is required for substrate recognition. A hinge region was identified that is critical for switching between the two conformations and was directly implicated in the high-affinity binding of GSK2830371 to PPM1D. We propose that the two conformations represent active and inactive forms of the protein reflected by the position of the flap, and that binding of GSK2830371 shifts the equilibrium to the inactive form. Finally, we found that C-terminal truncating mutations proximal to residue 400 result in destabilization of the protein via loss of a stabilizing N- and C-terminal interaction, consistent with the observation from human genetic data that nearly all PPM1D mutations in cancer are truncating and occur distal to residue 400. Taken together, our findings elucidate the mechanism by which binding of a small molecule to an allosteric site of PPM1D inhibits its activity and provides insights into the biology of PPM1D.

A comprehensive overview of PPM1A: From structure to disease.

PPM1A (magnesium-dependent phosphatase 1 A, also known as PP2Cα) is a member of the Ser/Thr protein phosphatase family. Protein phosphatases catalyze the removal of phosphate groups from proteins via hydrolysis, thus opposing the role of protein kinases. The PP2C family is generally considered a negative regulator in the eukaryotic stress response pathway. PPM1A can bind and dephosphorylate various proteins and is therefore involved in the regulation of a wide range of physiological processes. It plays a crucial role in transcriptional regulation, cell proliferation, and apoptosis and has been suggested to be closely related to the occurrence and development of cancers of the lung, bladder, and breast, amongst others. Moreover, it is closely related to certain autoimmune diseases and neurodegenerative diseases. In this review, we provide an insight into currently available knowledge of PPM1A, including its structure, biological function, involvement in signaling pathways, and association with diseases. Lastly, we discuss whether PPM1A could be targeted for therapy of certain human conditions.

Protein Phosphatase PP2C Identification in Entamoeba spp.

Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed.

Role of active site arginine residues in substrate recognition by PPM1A.

Reversible protein phosphorylation is a key mechanism for regulating numerous cellular events. The metal-dependent protein phosphatases (PPM) are a family of Ser/Thr phosphatases, which uniquely recognize their substrate as a monomeric enzyme. In the case of PPM1A, it has the capacity to dephosphorylate a variety of substrates containing different sequences, but it is not yet fully understood how it recognizes its substrates. Here we analyzed the role of Arg33 and Arg186, two residues near the active site, on the dephosphorylation activity of PPM1A. The results showed that both Arg residues were critical for enzymatic activity and docking-model analysis revealed that Arg186 is positioned to interact with the substrate phosphate group. In addition, our results suggest that which Arg residue plays a more significant role in the catalysis depends directly on the substrate.

A conserved allosteric element controls specificity and activity of functionally divergent PP2C phosphatases from Bacillus subtilis.

Reversible phosphorylation relies on highly regulated kinases and phosphatases that target specific substrates to control diverse cellular processes. Here, we address how protein phosphatase activity is directed to the correct substrates under the correct conditions. The serine/threonine phosphatase SpoIIE from Bacillus subtilis, a member of the widespread protein phosphatase 2C (PP2C) family of phosphatases, is activated by movement of a conserved α-helical element in the phosphatase domain to create the binding site for the metal cofactor. We hypothesized that this conformational switch could provide a general mechanism for control of diverse members of the PP2C family of phosphatases. The B. subtilis phosphatase RsbU responds to different signals, acts on a different substrates, and produces a more graded response than SpoIIE. Using an unbiased genetic screen, we isolated mutants in the α-helical switch region of RsbU that are constitutively active, indicating conservation of the switch mechanism. Using phosphatase activity assays with phosphoprotein substrates, we found that both phosphatases integrate substrate recognition with activating signals to control metal-cofactor binding and substrate dephosphorylation. This integrated control provides a mechanism for PP2C family of phosphatases to produce specific responses by acting on the correct substrates, under the appropriate conditions.

The ARF tumor suppressor targets PPM1G/PP2Cγ to counteract NF-κB transcription tuning cell survival and the inflammatory response.

Inducible transcriptional programs mediate the regulation of key biological processes and organismal functions. Despite their complexity, cells have evolved mechanisms to precisely control gene programs in response to environmental cues to regulate cell fate and maintain normal homeostasis. Upon stimulation with proinflammatory cytokines such as tumor necrosis factor-α (TNF), the master transcriptional regulator nuclear factor (NF)-κB utilizes the PPM1G/PP2Cγ phosphatase as a coactivator to normally induce inflammatory and cell survival programs. However, how PPM1G activity is precisely regulated to control NF-κB transcription magnitude and kinetics remains unknown. Here, we describe a mechanism by which the ARF tumor suppressor binds PPM1G to negatively regulate its coactivator function in the NF-κB circuit thereby promoting insult resolution. ARF becomes stabilized upon binding to PPM1G and forms a ternary protein complex with PPM1G and NF-κB at target gene promoters in a stimuli-dependent manner to provide tunable control of the NF-κB transcriptional program. Consistently, loss of ARF in colon epithelial cells leads to up-regulation of NF-κB antiapoptotic genes upon TNF stimulation and renders cells partially resistant to TNF-induced apoptosis in the presence of agents blocking the antiapoptotic program. Notably, patient tumor data analysis validates these findings by revealing that loss of ARF strongly correlates with sustained expression of inflammatory and cell survival programs. Collectively, we propose that PPM1G emerges as a therapeutic target in a variety of cancers arising from ARF epigenetic silencing, to loss of ARF function, as well as tumors bearing oncogenic NF-κB activation.

Identification of HN252 as a potent inhibitor of protein phosphatase PPM1B.

Protein phosphatase 1B (PPM1B), a member of metal-dependent protein serine/threonine phosphatase family, is involved in the regulation of several signalling pathways. However, our understanding of its substrate interaction and physiological functions is still largely limited. There is no reported PPM1B inhibitor to date. In this study, we identified HN252, a p-terphenyl derivative, as a potent PPM1B inhibitor (Ki  = 0.52 ± 0.06 µM). HN252 binding to PPM1B displayed remarkable and specific inhibition of PPM1B in both in vitro and ex vivo. With the aid of this small molecular inhibitor, we identified 30 proteins' serine/threonine phosphorylation as potential substrates of PPM1B, 5 of which were demonstrated by immunoprecipitation, including one known (CDK2) and 4 novel ones (AKT1, HSP90B, ß-catenin and BRCA1). Furthermore, GO and KEGG analysis of dramatically phosphorylated proteins by PPM1B inhibition indicated that PPM1B plays roles in the regulation of multiple cellular processes and signalling pathways, such as gene transcription, inflammatory regulation, ageing and tumorigenesis. Our work provides novel insights into further investigation of molecular mechanisms of PPM1B.

Epitope Analysis and Efficacy Evaluation of Phosphatase 2C (PP2C) DNA Vaccine Against Toxoplasma gondii Infection.

Toxoplasma gondii infects almost all warm-blooded animals and negatively affects the health of a wide range of these animals, including humans. Protein phosphatase 2C (PP2C) is a T. gondii protein secreted by rhoptry organelles during host cell invasion. However, very little is known about whether this protein can induce protective immunity against T. gondii. In this study, bioinformatics analysis of PP2C revealed some useful information in the context of anti-toxoplasmosis treatments and vaccine research. In addition, the PP2C gene was amplified, and a eukaryotic expression vector (pEGFP-PP2C) was successfully constructed to express PP2C. Finally, the constructed pEGFP-PP2C was injected into mice to evaluate whether it could induce immunoprotection. Compared with the control groups, we found that immunizations with the pEGFP-PP2C plasmid could elicit specific IgG antibodies and cytokines against T. gondii infection. The survival of mice immunized with the pEGFP-PP2C plasmid was significantly prolonged compared with that of the control group mice. Based on the ability of pEGFP-PP2C to induce specific immune responses against T. gondii, we propose that PP2C merits consideration as a potential vaccine candidate against toxoplasmosis.

Cellular Identification and In Silico Characterization of Protein Phosphatase 2C (PP2C) of Cryptosporidium parvum.

PURPOSE: Cryptosporidium parvum is an Apicomplexa parasite that causes watery diarrhea (cryptosporidiosis), especially in children and immunocompromised adults (the latter in a very severe form). No effective treatment exists against infection by this parasite. Phosphatases participate in the regulation of various cellular functions and are thus considered potential therapeutic targets in many diseases. The aim of the present study was to indirectly identify and in silico characterize a protein phosphatase 2C of C. parvum. METHODS: Western blot and indirect immunofluorescence microscopy were performed with a polyclonal antibody against Leishmania major PP2C. Possible cross-reactivity with LmPP2C was assessed by in silico sequence homology to analyze phylogenetic relationships between distinct C. parvum PP2Cs. In addition, another bioinformatics approach was used to predict the possible relationship and function of C. parvum PP2C in the regulation of several cellular processes. RESULTS: Western blotting showed a protein of approximately 72 kDa. With immunofluorescence, PP2C was localized in the nucleus of oocysts (with some additional labeling in the cytoplasm) and at the apical region of sporozoites. By aligning C. parvum PP2C with known ortholog sequences and carrying out PPI analysis, a determination could be made of the degree of conservation of these enzymes, their possible relationship, and their function in the regulation of several cellular processes associated with a likely nuclear location. CONCLUSION: Microscopic localization by immunofluorescence identified CpPP2C at the nucleus in oocysts and at the apical end of the sporozoite body. Hence, this enzyme could be associated with proteins that have an important role in the regulation of transcription and other processes orchestrated by MAPK kinases, according to in silico analysis.

Physiologically relevant orthogonal assays for the discovery of small-molecule modulators of WIP1 phosphatase in high-throughput screens.

WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.

Targeting of PP2Cδ By a Small Molecule C23 Inhibits High Glucose-Induced Breast Cancer Progression In Vivo.

Aims: Epidemiologic evidence indicates that diabetes may increase risk of breast cancer (BC) and mortality in patients with cancer. The pathophysiological relationships between diabetes and cancer are not fully understood, and personalized treatments for diabetes-associated BC are urgently needed. Results: We observed that high glucose (HG), via activation of nuclear phosphatase PP2Cδ, suppresses p53 function, and consequently promotes BC cell proliferation, migration, and invasion. PP2Cδ expression is higher in tumor tissues from BC patients with hyperglycemia than those with normoglycemia. The mechanisms underlying HG stimulation of PP2Cδ involve classical/novel protein kinase-C (PKC) activation and GSK3ß phosphorylation. Reactive oxygen species (ROS)/NF-κB pathway also mediates HG induction of PP2Cδ. Furthermore, we identified a 1,5-diheteroarylpenta-1,4-dien-3-one (Compound 23, or C23) as a novel potent PP2Cδ inhibitor with a striking cytotoxicity on MCF-7 cells through cell-based screening assay for growth inhibition and activity of a group of curcumin mimics. Beside directly inhibiting PP2Cδ activity, C23 blocks HG induction of PP2Cδ expression via heat shock protein 27 (HSP27) induction and subsequent ablation of ROS/NF-κB activation. C23 can thus significantly block HG-triggered inhibition of p53 activity, leading to the inhibition of cancer cell proliferation, migration, and invasion. In addition, hyperglycemia promotes BC development in diabetic nude mice, and C23 inhibits the xenografted BC tumor growth. Conclusions and Innovation: Our findings elucidate mechanisms that may have contributed to diabetes-associated BC progression, and provide the first evidence to support the possible alternative therapeutic approach to BC patients with diabetes. Antioxid. Redox Signal. 30, 1983-1998.

Crystal structure and catalytic activity of the PPM1K N94K mutant.

Protein Phosphatase Mg2+ /Mn2+ -Dependent 1K (PPM1K),also named as PP2Cm or branched-chain α-ketoacid dehydrogenase complex phosphatase, is a member of the metal-dependent phosphatase family and an important metabolic regulator. Single nucleotide polymorphisms (SNPs) in PPM1K contributing to protein functional defects have been found to be associated with numerous human diseases, such as cardiovascular disease, maple syrup urine disease, type 2 diabetes, and neurological disease. PPM1K N94K is an identified missense mutant produced by one of the SNPs in the human PPM1K coding sequence. However, the effects of the N94K mutant on its activity and structural property have not been defined. Here, we performed a detailed enzymological study using steady-state kinetics in the presence of pNPP or phospho-peptide substrates and crystallographic analyses of the wild-type and N94K PPM1K. The PPM1K-N94K significantly impaired its Mg2+ -dependent catalytic activity and structural analysis demonstrated that the N94K mutation induced a conformational change in the key residue in coordinating the Mg2+ in the active site. Specifically, three Mg2+ were located in the active site of the PPM1K N94K instead of two Mg2+ in the PPM1K wild type. Therefore, our results provide a structure basis for the metal ion-dependent PPM1K-N94K phosphatase activity.

Isolation of a novel protein phosphatase2C in rice and its response to gibberellin.

Protein phosphatase 2Cs (PP2Cs) have been referred to act as negative modulators of the protein kinase pathways involved in different environmental stress responses and developmental processes. In Arabidopsis, PP2Cs have been extensively studied and some are known to negatively regulate abscisic acid signaling. In rice, PP2Cs are scarcely characterized functionally. Here, we identified a novel PP2C from rice (OsPP2C34), which is highly inducible by gibberellin (GA) and expressed in various tissues. Subcellular localization analysis in maize protoplasts using a green fluorescence protein fusion vector localized OsPP2C34 to the cytosol. Genetic analysis of T-DNA insertional mutants revealed that plant height and internode length were significantly shorter in mutants than in corresponding wild types under GA treatment. The induction of the GA-inducibleα-amylase genes RAmy3E and OsAmy was delayed in mutant plants. The substrate of OsPP2C34 was identified by immunoblotting using anti serine/threonine antibodies. A 65 kDa protein was phosphorylated in Ospp2c34-1 but dephosphorylated in the wild type during early germination stage. Overall, the present results indicated that OsPP2C34 is involved inα-amylase expression of GA signal transduction pathway.

Leishmania donovani PP2C: Kinetics, structural attributes and in vitro immune response.

Most of the signaling pathways are regulated by reversible phosphorylation-dephosphorylation which involves enzymes- kinases and phosphatases. Current knowledge about the protein phosphatases in parasites like Trypanosoma and Leishmania is very minimal despite their enormousity. In present study, full length ORF of Leishmania donovani PP2C was cloned into expression vector followed by purification and molecular weight determination using Ni-NTA affinity and gel giltration chromatography respectively. Purified LdPP2C was found to be enzymatically active, while inhibition study suggested that sanguinarine acts as a non-competitive inhibitor. CD and fluorescence spectroscopy results indicated towards an adequate protein conformation from pH 3.5 to 8.5. The quenching constant (Ksv) and free energy (ΔG) of LdPP2C was found to be 11.1 ±â€¯0.2 mM-1 and 2.0 ±â€¯1.1 kcal mol-1 in presence of acrylamide and urea respectively. The protein was found to elicit the innate immune functions through upregulation of pro-inflammatory cytokines (TNF-α and IL-6) as well as nitric oxide generation. Simultaneously, these cytokines were found to be fairly higher in protein treated cells as compared to untreated cells at transcript level too. These observations advocate that LdPP2C generates a pro-inflammatory environment in macrophages and hence plays important role in immunomodulation. Computational modelling showed similar three-dimensional structure and metal binding sites present in other member of PP2C subfamily, while docking studies revealed its interaction with substrate as well as its specific inhibitor. Our study has provided first time reports on enzyme kinetics, structural features and immune response inside the host macrophage of metal-dependent protein phosphatases from a trypanosomatid parasite.

The Molecular Mechanism of Alpha-Synuclein Dependent Regulation of Protein Phosphatase 2A Activity.

BACKGROUND/AIMS: Alpha-synuclein (α-Syn) is a neuronal protein that is highly implicated in Parkinson's disease (PD), and protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase that is associated with neurodegenerative diseases, such as PD. α-Syn can directly upregulate PP2A activity, but the underling mechanism remains unclear. Therefore, we investigated the molecular mechanism of α-Syn regulating PP2A activity. METHODS: α-Syn and its truncations were expressed in E.coli, and purified by affinity chromatography. PP2A Cα and its mutants were expressed in recombinant baculovirus, and purified by affinity chromatography combined with gel filtration chromatography. The interaction between α-Syn and PP2A Cα was detected by GST pull-down assay. PP2A activity was investigated by the colorimetric assay. RESULTS: The hydrophobic non-amyloid component (NAC) domain of α-Syn interacted with PP2A Cα and upregulated its activity. α-Syn aggregates reduced its ability to upregulate PP2A activity, since the hydrophobic domain of α-Syn was blocked during aggregation. Furthermore, in the hydrophobic center of PP2A Cα, the residue of I123 was responsible for PP2A to interact with α-Syn, and its hydrophilic mutation blocked its interaction with α-Syn as well as its activity upregulation by α-Syn. CONCLUSIONS: α-Syn bound to PP2A Cα by the hydrophobic interaction and upregulated its activity. Blocking the hydrophobic domain of α-Syn or hydrophilic mutation on the residue I123 in PP2A Cα all reduced PP2A activity upregulation by α-Syn. Overall, we explored the mechanism of α-Syn regulating PP2A activity, which might offer much insight into the basis underlying PD pathogenesis.

A trapped human PPM1A-phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase 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 Mg2+ or Mn2+ ions for activity in vitro The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ 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, PPM1Acat, 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 PPM1Acat 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 PPM1Acat toward a phosphopeptide substrate supported a random-order, 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.

Functions and dysfunctions of Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) and CaMKP-N/PPM1E.

Intracellular signal transduction is built on the basis of the subtle balance between phosphorylation and dephosphorylation. Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F/POPX2) and CaMKP-N (PPM1E/POPX1) are Ser/Thr phosphatases that belong to the PPM (protein phosphatase, Mg2+/Mn2+-dependent) family. The former was discovered in rat brain as a novel protein phosphatase regulating Ca2+/calmodulin-dependent protein kinases (CaMKs), whereas the latter was first identified in human cDNA databases using the rat CaMKP sequence. Subsequent studies have revealed that they are involved in various cellular functions through regulation of not only CaMKs but also other protein kinases such as AMP-activated protein kinase. Furthermore, accumulating evidence shows possible involvement of CaMKP and CaMKP-N in the pathogenesis of various diseases including cancer. Therefore, the biochemistry of CaMKP and CaMKP-N largely contributes to molecular medicine targeting these phosphatases. In this review, we summarized recent progress in the enzymology and biology of CaMKP and CaMKP-N. We also focused on etiology studies in which CaMKP and CaMKP-N are involved. Based on the emerging evidence, future perspectives of studies on these phosphatases and related issues to be elucidated are discussed.

Modulation of ABA Signaling by Altering VxGΦL Motif of PP2Cs in Oryza sativa.

The abscisic acid (ABA) signaling pathway is regulated by clade A type 2C protein phosphatases (PP2CAs) in plants. In the presence of ABA, PP2Cs release stress/ABA-activated protein kinases by binding to ABA-bound receptors (PYL/RCARs) for activation. Although the wedging tryptophan in PP2Cs is critical in the interaction with PYL/RCARs in Arabidopsis and rice, it remains elusive as to how other interface regions are involved in the interaction. Here, we report the identification of a conserved region on PP2Cs, termed the VxGΦL motif, which modulates the interaction with PYL/RCARs through its second and fourth residues. The effects of the second and fourth residues on the interaction of OsPP2C50 with several OsPYL/RCAR proteins were investigated by systematic mutagenesis. One OsPP2C50 mutant, VFGML ("FM") mutant, lowered the affinity to OsPYL/RCAR3 by ∼15-fold in comparison with the wild-type. Comparison of the crystal structures of wild-type OsPP2C50:ABA:OsPYL/RCAR3 with those composed of FM mutant revealed local conformational changes near the VxGΦL motif, further supported by hydrogen-deuterium exchange mass spectrometry. In rice protoplasts, ABA signaling was altered by mutations in the VxGΦL motif. Transgenic Arabidopsis plants overexpressing OsPP2C50 and OsPP2C50FM showed altered ABA sensitivity. Taken together, the VxGΦL motif of PP2Cs appears to modulate the affinity of PP2Cs with PYL/RCARs and thus likely to alter the ABA signaling, leading to the differential sensitivity to ABA in planta.

Conformational Changes in Active and Inactive States of Human PP2Cα Characterized by Hydrogen/Deuterium Exchange-Mass Spectrometry.

PPM serine/threonine protein phosphatases function in signaling pathways and require millimolar concentrations of Mn2+ or Mg2+ ions for activity. Whereas the crystal structure of human PP2Cα displayed two tightly bound Mn2+ ions in the active site, recent investigations of PPM phosphatases have characterized the binding of a third, catalytically essential metal ion. The binding of the third Mg2+ to PP2Cα was reported to have millimolar affinity and to be entropically driven, suggesting it may be structurally and catalytically important. Here, we report the use of hydrogen/deuterium exchange-mass spectrometry and molecular dynamics to characterize conformational changes in PP2Cα between the active and inactive states. In the presence of millimolar concentrations of Mg2+, metal-coordinating residues in the PP2Cα active site are maintained in a more rigid state over the catalytically relevant time scale of 30-300 s. Submillimolar Mg2+ concentrations or introduction of the D146A mutation increased the conformational mobility in the Flap subdomain and in buttressing helices α1 and α2. Residues 192-200, located in the Flap subdomain, exhibited the greatest interplay between effects of Mg2+ concentration and the D146A mutation. Molecular dynamics simulations suggest that the presence of the third metal ion and the D146A mutation each produce distinct conformational realignments in the Flap subdomain. These observations suggest that the binding of Mg2+ to the D146/D239 binding site stabilizes the conformation of the active site and the Flap subdomain.