Formation of ternary receptor/ligand-phosphatase complexes.

<p>Both the PYL1 and PYL2 receptors were able to interact with the HAB1 and ABI2 phosphatases to form complexes in the presence of (+)-ABA. Melt curves of ligand-bound receptors (dashed blue lines), PP2Cs (dashed red lines), and receptor-PP2C complexes (solid green lines). Receptors and PP2C were at 10 µM, (+)-ABA at 500 µM. (<b>A</b>) PYL1 with ABI2 (left) and HAB1 (right). (<b>B</b>) PYL2 with ABI2 (left) and HAB1 (right).</p

Crystal structure of HAB1.

<p>Ribbon representation of the HAB1 phosphatase domain (PDB: 4LA7) with C186 and C274 presented as stick models and the remaining cysteine residues as yellow patches. The SnRK2.6 interacting site is shown in magenta and MgCl₂ ions as magenta spheres (figure drawn from).</p

H₂O₂ inhibits the direct interaction of HAB1 with SnRK2.6 and PYR1.

<p>A, B) AlphaScreen assays showing interactions between His₆-MBP-HAB1(171–511 aa) wild type, C186/274S double mutant and R505A mutant protein and biotin-SnRK2.6(11–362 aa), biotin-SnRK2.6 ABA box peptide and biotin-PYR1(9–182 aa) with 10 µM ABA in the absence or presence of 0.3 mM (A) or 1 mM H₂O₂ (B). Controls are HAB1 proteins in the absence of SnRK2.6 or PYR1 as well as SnR2.6 or PYR1 in the absence of HAB1 (n = 3, error bar represent s.d.). C) Non-reducing SDS PAGE of untreated wild type HAB1 (−) and HAB1 treated with 1 mM of H₂O₂ (+).</p

Stoichiometry of receptor/ligand-phosphatase complexes.

<p>Transition curves of ABA-bound PYL1 and PYL2 in the presence of increasing amounts of HAB1 and ABI1 PP2Cs. The melt curves for receptors in the absence of PP2Cs (dashed grey lines ) and PP2Cs in the absence of receptors (dashed black lines ) are shown as references. The positions of uncomplexed, excess receptors and PP2Cs are indicated by arrows. Each panel shows titrations of 10 µM receptor with PP2Cs at 1 µM (pale yellow line), 5 µM (yellow line), 10 µM (blue line) and 20 µM (orange line) PP2C. (<b>A</b>) PYL1/ABA titrated with HAB1 (left) and ABI2 (right). (<b>B</b>) PYL2 titrated with HAB1 (left) and ABI1 (right).</p

H₂O₂ induces formation of HAB1 dimers that are catalytically compromised.

<p>A, B, upper panels: Gel filtration profiles of HAB1 wildtype and C186S/C274S mutant protein in the presence and absence of H₂O₂. Wildtype His₆-MBP-HAB1 elutes as monomers in the absence of H₂O₂ (blue chromatogram) and as a mixture of dimers (at 60 ml elution volume) and monomers (at 70 ml) upon treatment with 0.3 mM H₂O₂ (red chromatogram). In contrast, His₆-MBP-HAB1 C186S/C274S elutes exclusively as monomers. *MW: molecular weight standards; mAU: absorbance at 280 nm×1000. Lower panels: Reducing and non-reducing SDS PAGE of gel filtration fractions of wild type and C186S/C274S HAB1 proteins, respectively. C) Phosphatase activity of HAB1 protein from the untreated monomer fraction (no H₂O₂), from the dimer and monomer fractions of wild type HAB1 treated with 0.3 mM H₂O₂, and from the monomer fraction of HAB1 C186S/C274S treated with 0.3 mM H₂O₂.</p

Sequence alignment of HAB1, ABI2 and ABI1.

<p>Cysteines in the phosphatase domain of HAB1 are indicated by black boxes and asterisks. C186 and C274 are highlighted by red asterisks. The numbers on top of the alignment indicate amino acid positions within HAB1.</p

PYL1/PP2C and PYL2/PP2C radioligand competition assays.

<p>Competition of the interactions between ³H-ABA and the PYL1/HAB1 (<b>A</b>) and PYL2/HAB1 (<b>B</b>) coreceptor complexes by ABA and pyrabactin (n = 3, error bars represent s.d.).</p

H₂O₂ prevents the HAB1-mediated inhibition of SnRK2.6 kinase activity.

<p>A) SnRK2.6 trans-phosphorylation activity at high (30∶50 molar) HAB1:SnRK2.6 ratio. B) SnRK2.6 auto- and trans-phosphorylation activity at low (30∶1000 molar) HAB1:SnRK2.6 ratio. Recombinant SnRK2.6 was incubated at the indicated concentrations with a fragment of the transcription factor ABF2 [GST-ABF2(73–120 aa)] and with ³²P-γATP in the presence and absence of H₂O₂-treated wild type (wt) and mutant (mt; C186S/C274S) HAB1.</p

Reversible oxidation of HAB1 by H₂O₂.

<p>A) Sensitivity of full length (1–511 aa) and phosphatase domain (171–511 aa) of HAB1 to H₂O₂ treatment. B) Reactivation of H₂O₂ treated HAB1 by reducing agents (n = 3, error bar represent s.d.).</p

Schematic representation of thermostability profiles corresponding to the various aspects of the signaling pathway.

<p>(<b>A</b>) Stabilization of receptor by ligand-binding is demonstrated by the shift in the melt curve, and ligand-induced protein-protein interaction results in the formation of a more stable complex that unfolds cooperatively with a Tm higher than that of both individual proteins. (<b>B</b>) A SnRK2-MBP fusion protein exhibits a bi-phasic melt curve, of which only the transition curve corresponding to untagged SnRK2 shifts in the presence of SnRK2 inhibitors.</p