AlphaFold 3-Assisted Deciphering of the DNA Recognition by DREB1 Transcription Factors in Rice.

Rice genome encodes ten OsDREB1 proteins that regulate tolerance to abiotic stresses such as cold and drought. OsDREB1s can bind to the C-repeat (CRT) element, dehydration response element (DRE), and GCC-box in gene promoters for transcription regulation. However, the recognition mechanism of OsDREB1s to these DNA elements remains unclear. Here, the structures of OsDREB1s were modelled using AlphaFold 3, which revealed a typical AP2 domain and a disordered KRP/RAGR motif adjacent to AP2 in all OsDREB1s. Structure modeling of OsDREB1A binding to CRT, DRE, and GCC-box showed that four Arg residues and a Glu (E66) from AP2 play important roles in binding to the major groove of DNA, while R40 in the KRP/RAGR motif was predicted to interact with the minor groove. The structure models revealed a few differences in the binding details for CRT, DRE, and GCC-box. Consistent with these predictions, OsDREB1A was evidenced to bind with the three DNA elements in slightly different affinities through EMSA experiments. Mutation analysis verified the key role of R40 and E66 in binding to CRT. Considering the highly conserved structure and sequence of the KRP/RAGR motif and AP2, we speculate that the DNA recognition mechanism found for OsDREB1A may be common for all OsDREB1s.