ABA-regulated Protein Phosphatase 2C

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Five plant protein phosphatase 2Cs - 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 3ujk Middle panel- HAB1 (gold), with Mg2+ and SO42- in complex with SnRK2.6 (blue) 3ujg Right panel - ABI2 bound to PYL2.ABA 3ujl

3ujk - ABI2


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


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


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.















ABA-regulated PP2C structures

3jrq, 3kdj, 3nmn – AtPP2C (ABI1) + Pyl1 – Arabidopsis thaliana
3nmt, 3kb3, 3ujl – AtPP2C (HAB1)+ Pyl2
3nmv, , 3ujl - AtPP2C (ABI2)+ Pyl2
4ds8 – AtPP2C (HAB1) + Pyl3 + Mn
3rt0 – AtPP2C (mutant) (HAB1) + Pyl10
3qn1, 3zvu – AtPP2C (HAB1)+ Pyr1
3ujg – AtPP2C (HAB1)+ SNRK2
3ujk – AtPP2C (ABI2)

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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Alice Harmon

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