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

Mazur, Sharlyn J.; Gallagher, Elyssia S.; Debnath, Subrata; Durell, Stewart R.; Anderson, Kyle W.; Jenkins, Lisa M. Miller; Appella, Ettore; Hudgens, Jeffrey W.

doi:10.1021/acs.biochem.6b01220

Cited by 6 publications

(11 citation statements)

References 50 publications

(240 reference statements)

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“…This mechanism is supported by the structure of the complex, which indicates that both the D146/D239 and D239/D243 subsites are accessible to ions in solution in the presence of bound substrate, and by solution studies suggesting that the binding of either Mg 2+ or substrate results in reduced conformational mobility (30). Although the substrate can bind in the absence of the weaklybound metal ion, the binding affinity is considerably higher for the metal-bound state.…”

Section: Discussionmentioning

confidence: 76%

“…Recently, we used HDX-MS to characterize the conformational mobility of full-length PPM1A and PPM1A D146A under conditions of low (0.1 mM) and moderate (2 mM) Mg 2+ concentrations (30). The Flap subdomain was identified as a conformationally mobile region in which amide hydrogen-deuterium exchange was affected both by Mg 2+ concentrations and the identity of the residue at position 146.…”

Section: Small-angle X-ray Scattering and Molecular Dynamicsmentioning

confidence: 99%

“…D239 coordinates M1 in addition to its involvement in both M3 subsites; D239 mutants are inactive. We used hydrogen-deuterium exchangemass spectrometry (HDX-MS) to investigate the conformational mobility of PPM1A in the presence of low or moderate magnesium ion concentrations (30). We found that the binding of Mg 2+ to the catalytically essential D146/D239 subsite restricted conformational mobility of the active site and specific regions of the Flap subdomain, suggesting that the third metal ion stabilizes the native structure of the enzyme (30).…”

Section: Introductionmentioning

confidence: 99%

“…We used hydrogen-deuterium exchangemass spectrometry (HDX-MS) to investigate the conformational mobility of PPM1A in the presence of low or moderate magnesium ion concentrations (30). We found that the binding of Mg 2+ to the catalytically essential D146/D239 subsite restricted conformational mobility of the active site and specific regions of the Flap subdomain, suggesting that the third metal ion stabilizes the native structure of the enzyme (30). These studies support the idea that conformational stabilization of the Flap, which is critical for specific substrate recognition, may be coupled to catalytic activity through the binding of the third metal ion.…”

Section: Introductionmentioning

confidence: 99%

“…As the initiating methionine is absent in mature, endogenous PPM1A and existing crystal structures begin with Gly2, the additional amino acids may interfere with crystallization. Moreover, HDX-MS experiments suggested increased conformational mobility of PPM1A D146A compared with the WT enzyme (30). To address these concerns, we reverted the Nterminus to the endogenous form, introduced the more conservative Asp to Glu mutation at position 146, and removed helices α7, α8, and α9 to produce the WT PPM1A cat and PPM1A cat D146E variants of the catalytic domain (aa 2-297).…”

Section: Introductionmentioning

confidence: 99%

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A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

Debnath

Košek²,

Tagad

et al. 2018

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 76%

Section: Small-angle X-ray Scattering and Molecular Dynamicsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

Debnath

Košek²,

Tagad

et al. 2018

Journal of Biological Chemistry

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

Bradshaw

2021

Journal of Biological Chemistry

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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.

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Automated Removal of Phospholipids from Membrane Proteins for H/D Exchange Mass Spectrometry Workflows

2018

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Membrane proteins are currently the most common targets for pharmaceuticals. However, characterization of their structural dynamics by hydrogen/deuterium exchange mass spectrometry (HDX-MS) is sparse due to insufficient automated methods to handle full-length membrane proteins in lipid bilayers. Additionally, membrane lipids used to mimic the membrane environment and to solubilize membrane proteins can impair chromatography performance and cause ion suppression in the mass spectrometer. The workflow discussed herein advances HDX-MS capabilities and other MS applications for membrane proteins by providing a fully automated method for HDX-MS analysis based on a phospholipid removal scheme compatible with robotic handling. Phospholipids were depleted from protein samples by the addition of zirconium oxide beads, which were subsequently removed by inline filtration using syringeless nanofilters. To demonstrate this method, single-pass transmembrane protein FcγRIIa (CD32a) expressed into liposomes was used. Successful depletion of phospholipids ensured optimal liquid-chromatography-mass-spectrometry performance, and measurement of peptides from the transmembrane domain of FcγRIIa indicated phospholipids associated with this region were either not present or did not shield the transmembrane domain from digestion by pepsin. Furthermore, amino acid sequence coverage provided by this method was suitable to enable future measurement of structural dynamics of ectodomain, transmembrane domain, and endodomain of FcγRIIa. Moreover, this method is the first to enable fully automated HDX-MS on full-length transmembrane proteins in lipid bilayers, a notable advancement to facilitate understanding of membrane proteins, development of pharmaceuticals, and characterization for regulatory agencies.

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

Cited by 6 publications

References 50 publications

A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

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

Automated Removal of Phospholipids from Membrane Proteins for H/D Exchange Mass Spectrometry Workflows

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