Protein Phosphatase 2C

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Contents

Function

  • Plant protein phosphatase 2C

Five plant protein phosphatase 2Cs or protein serine-threonine phosphatase or protein phosphatase 1A - ABI1, ABI2, HAB1, HAB2, and PP2CA/AHG3 - are essential components of the core ABA Signaling Pathway[1][2][3]. In the absence of ABA, the PP2C forms a complex with a SnRK2 protein kinase and inactivates it. In the presence of ABA, the PP2C is bound instead to the ABA-bound ABA receptor. This frees the kinase to phosphorylate its substrates.

Below are structures of ABI2 monomer, HAB1 in complex with an ABA receptor (PYL2) and of ABI2 in complex with SNRK2.6. The PP2C binds its partners via an interface that includes a tryptophan (W290 of ABI2; W385 in HAB1) and residues near the active site. In comparison ABI2, HAB1 has a longer N-terminal sequence and shorter C-terminal sequence, and it has two Mg2+-binding sites, instead of three.

Protein phosphatase

Left panel - ABI2 complex with Mg2 ions (green)3ujk Middle panel- HAB1 (gold), with Mg2+ and SO42- in complex with SnRK2.6 (blue) 3ujg Right panel - ABI2 bound to PYL2., abscicic acid and with Mg2 3ujl

3ujk - ABI2

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3ujk scenes
PP2Cs have two central antiparallel beta sheets that are flanked by alpha helices. Three magnesium ions (green spheres) occupy the active site of this structure.
is located at one end of the beta sheet sandwich. The arginine residue that binds the phosphate removed from the substrate is shown in blue ball and stick. Tryptophan 290, which binds to the ABA receptor or to SNRK2 protein kinase, is shown in gold ball and stick.
are coordinated by oxygen atoms from water (small red spheres) and side chains of conserved glutamate and aspartate residues (CPK ball and stick). An unconserved methionine residue in ABI2 (contains yellow sulfur atom) also binds a magnesium ion. A water molecule that bridges the magnesium ions is thought the nucleophile in the SN2 dephosphorylation mechanism.












3ujg - SnRK2.6-HAB1

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3ujg scenes
The two enzymes are bound via interface their active sites. The phosphatase inactivates the kinase by dephosphorylating the kinase activation loop and by sterically blocking the kinase active site. The complex was constructed as a fusion protein with a 6His-tag at the N-terminus of SnRK2.6 (residues 11–362) fused to HAB1(172–511) via a GSGSAGSAAGS linker. Mutations of D296A and E297A in SnRK2.6 were introduced at the crystal packing interface to reduce surface entropy.
The activation loop (blue trace) of SnRK2.6 is inserted into the catalytic site (marked by the magnesium ions) of the phosphatase. The phosphorylatable residue of the activation loop S175 (sidechain in CPK ball and stick) is positioned near the magnesium ions. Arginine 199 (equivalent of Arg 122 in ABI1) is shown in Cyan ball and stick. Residues of the phosphatase interact with kinase structures that are critical for activity. Tryptophan 385 of HAB1 (equivalent of Try 290 of ABI1) is shown in gold ball and stick. It interacts with residues Arg 139 and Glu 144 (CPK ball and stick) of the catalytic loop (orchid) and Ile 183 of the activation loop (blue).
SnRK2.6 is shown in blue cartoon, and HAB1 in gold spacefill. The ABA box sequence in the C-terminus of the kinase is is not resolved, but it would extend from the C-terminal end of the SNRK2 box helix (cyan helix). It is proposed that the ABA box sequence, which is highly acidic, binds to a patch of basic residues (blue) on the surface of the phosphatase.[4]

3ujl - PYL2-ABI2

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3ujl scenes
Complex between the protein phosphatase 2C ABI2 (gold) and the ABA receptor PYL2 (blue). ABA is shown in CPK spheres). Magnesium ions in the active site of ABI2 are shown as green spheres.
The gate (orchid) and latch (blue) loops of the ABA receptor are closed over the bound ABA (CPK spheres). The gate residue proline 92 (orchid ball and stick) interacts with tryptophan 290 of ABI2 (gold ball and stick) and with residues in a hydrophobic loop (dark red trace) of the phosphatase. The gate also interacts with residues surrounding the phosphatase's active site, which is marked by magnesium ions (green spheres). Gate residue serine 89 (side chain in CPK ball and stick) is in close proximity to arginine 122 of the phosphatase (cyan ball and stick), which binds the phosphate of substrates.















  • Human protein phosphatase 2C

Quartenary structure of human protein phosphatase PP2Cm with Mg(II) (PDB ID 4DA1)

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Protein Phosphatases 2C are essencial enzymes involved in the regulation of several signaling pathways of branched-chain α-ketoacid dehydrogenase complex (BCKDC) by phosphorylation/dephosphorylation. The PP2C Family are Mg2+ and Mn2+ dependent monomeric proteins with two characteristic structural domains: a catalytic domain N-terminal with six alpha-helices, and a C-terminal region with three alpha-helices. The multienzyme complex uses numerous copies of three enzymes as major building blocks E1, E2 and E3. A dihydrolipoyl transacylase (E2) forms the core of the complex with 24 copies in octahedral symmetry.

The human branched-chain α-ketoacid dehydrogenase complex ser/thr phosphatase, PP2Cm, (BDP) is attached to the E2 core through non-covalent bonds. PP2Cm is distinguished from other groups of phosphatases by its structural distinction, absolute requirement for divalent cation, the catalytic domain and shows Mn2+/Mg2+ dependent phosphatase activity. PP2Cm structure has two central antiparallel beta sheets that are flanked by alpha helices and the is located at one end of the beta-sheet sandwich containing two coordenated by residues. At high levels of branched-chain ketoacids PP2Cm dephosphorylates Ser-337 and activates mitochondrial BCKDC complex by associating with the E2 component of the complex. The water molecules at the binuclear metal centre coordinate the phosphate group of the substrate, each ion is hexa-coordinated by from water, providing a nucleophile and general acid in the dephosphorylation reaction, and Arg33 creates a local positive electrostatic potential on the protein for recognition of the phosphate group of the substrate. The nucleophile is the metal-bridging water molecule which could attack the phosphorus atom in an SN2 mechanism. Coordination to two Mg2+ ions may stabilize the morenucleophilic hydroxide ion species. Other ions such as Ca2+, Zn2+ and Ni2+ inactivate the enzyme by competitively inhibiting Mn2+ or Mg2+ binding.

3D Structures of protein phosphatase 2C

See Protein phosphatase 3D structures

References

  1. Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E. Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science. 2009 May 22;324(5930):1064-8. doi: 10.1126/science.1172408. Epub 2009, Apr 30. PMID:19407143 doi:10.1126/science.1172408
  2. Park SY, Fung P, Nishimura N, Jensen DR, Fujii H, Zhao Y, Lumba S, Santiago J, Rodrigues A, Chow TF, Alfred SE, Bonetta D, Finkelstein R, Provart NJ, Desveaux D, Rodriguez PL, McCourt P, Zhu JK, Schroeder JI, Volkman BF, Cutler SR. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science. 2009 May 22;324(5930):1068-71. doi: 10.1126/science.1173041. Epub 2009, Apr 30. PMID:19407142 doi:10.1126/science.1173041
  3. Yoshida T, Nishimura N, Kitahata N, Kuromori T, Ito T, Asami T, Shinozaki K, Hirayama T. ABA-hypersensitive germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs. Plant Physiol. 2006 Jan;140(1):115-26. Epub 2005 Dec 9. PMID:16339800 doi:10.1104/pp.105.070128
  4. Soon FF, Ng LM, Zhou XE, West GM, Kovach A, Tan MH, Suino-Powell KM, He Y, Xu Y, Chalmers MJ, Brunzelle JS, Zhang H, Yang H, Jiang H, Li J, Yong EL, Cutler S, Zhu JK, Griffin PR, Melcher K, Xu HE. Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases. Science. 2012 Jan 6;335(6064):85-8. Epub 2011 Nov 24. PMID:22116026 doi:10.1126/science.1215106

See Also

[1] Abscisic Acid in Wikipedia

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