PYL2, PYL1, and HAB1 were expressed as H6-GST or H6Sumo fusion proteins in E. coli. Proteins were purified by Ni-NTA chromatography, followed by proteolytic release of tags and size-exclusion chromatography. For formation of PYL2-ABA and HAB1-PYL2-ABA complexes, ABA was mixed with PYL2 and HAB1-PYL2 at 5:1 ratios. Crystals were grown by vapor diffusion and diffraction data were collected from cryo-protected crystals at beamlines 21-ID-D and 21-ID–F at the Advanced Photon Source at Argonne National Laboratories. Structures were solved by molecular replacement in PHASER 26 (link) using the structure of the plant START protein Bet v 1 as model for PYL2 and the structure of the human PP2C PPM1B as model for HAB1. Models were manually fitted using O and Coot 27 (link),28 (link) and further refined using CNS and Refmac5 29 (link),30 (link).
Mutant proteins were expressed as H6GST-fusion proteins and purified by glutathione sepharose chromatography. Protein-protein interactions were determined by luminescence proximity AlphaScreen assay and by yeast two-hybrid assay. Biotinylated HAB1 for the luminescence proximity assay was generated by in vivo biotinylation of an avitag-HAB1 fusion protein. ABA binding was determined by scintillation proximity assay using 3H-labelled ABA. HAB1 phosphatase activity was measured by phosphate release from a SnRK2.6 phosphoprotein (Fig.1 -5 ) or from a generic pNPP phosphate substrate (Fig. 6b ).
For transgenic studies, wildtype and mutant 35S::GFP-PYR1 constructs were transformed by the floral dip method into pyr1/pyl1/pyl2/pyl3 quadruple mutants. Mutant complementation of GFP+ seedlings was assayed by root length measurements. The ABA signal transduction pathway was reconstituted in protoplasts by transient transfection of PYL2, PP2C, SnRK2.6, and ABF2 expression plasmids. Activation of an ABA-inducible CBF3promoter-LUC reporter by PYL2 mutant proteins was determined by luciferase assays normalized for β-glucuronidase activity from a UQ10-GUS reporter.Full Methods accompany this paper at www.nature.com/nature .
Mutant proteins were expressed as H6GST-fusion proteins and purified by glutathione sepharose chromatography. Protein-protein interactions were determined by luminescence proximity AlphaScreen assay and by yeast two-hybrid assay. Biotinylated HAB1 for the luminescence proximity assay was generated by in vivo biotinylation of an avitag-HAB1 fusion protein. ABA binding was determined by scintillation proximity assay using 3H-labelled ABA. HAB1 phosphatase activity was measured by phosphate release from a SnRK2.6 phosphoprotein (Fig.
For transgenic studies, wildtype and mutant 35S::GFP-PYR1 constructs were transformed by the floral dip method into pyr1/pyl1/pyl2/pyl3 quadruple mutants. Mutant complementation of GFP+ seedlings was assayed by root length measurements. The ABA signal transduction pathway was reconstituted in protoplasts by transient transfection of PYL2, PP2C, SnRK2.6, and ABF2 expression plasmids. Activation of an ABA-inducible CBF3promoter-LUC reporter by PYL2 mutant proteins was determined by luciferase assays normalized for β-glucuronidase activity from a UQ10-GUS reporter.
