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 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

Cartoon representation of the regulation of HAB1 by H₂O₂ during water stress.

<p>See text for details.</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

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

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