Fig. 1: Evolution, interactions, and structure of RCD1 and DREB2s guide selection of DREB2A variants with varying residual helicity.
a Phylogenetic relationship of the plant kingdom with land plants divided into non-vascular (dark green) and vascular plants, which are further split into seedless (green) and seed plants, finally split into non-flowering gymnosperms (light green) and flowering angiosperms (lightest green). Species are listed in color corresponding to evolution. The Arabidopsis thaliana interactome of RCD1 obtained from the STRING database is highlighted to the right with DREB2s indicated. b Multiple sequence alignment of the RCD1-RST binding region of DREB2A homologs from different plant species. The aligned region corresponds to Arabidopsis thaliana DREB2A(255-272) (AT5G05410) with the RCD1-RST binding region underlined. Based on α-helix (%) predicted by Agadir, the sequences were assigned to three groups: I: 14–19%, II: 2-8%, and III: <2% (blue-colored horizontal bars at left). The α-helical region of free and RCD1-RST-bound DREB2A are marked by the dashed box and the black bar above the alignment, respectively30. The position of the hydrophobic stable motif is outlined at top with key stabilizing residues of the Ncap boxed in gray and numbering according to Ncap. Residues in the helical region are colored according to predicted α-helix stabilizing (dark blue) or destabilizing (light blue) effect based on Agadir predictions. SLiM residues are marked with black asterisks. Helicity predictions by Agadir of variants at positions R266 (c) and D267 (d) in DREB2A with DREB2A-WT in black. All nonaromatic amino acids are included (gray). The substitutions selected for experimental characterization are colored, and DREB2A-D267R is shown in purple. (e) HADDOCK model of DREB2A(255-272) (orange) in complex with RCD1-RST(499-572) (silver, PDB: 5oao)25 with helix 1-4 (H1-H4) indicated. Left zoom shows surface exposure of D267 and R266 in DREB2A.
