Molecular switching in transcription through splicing and proline-isomerization regulates stress responses in plants

Abstract

The Arabidopsis thaliana DREB2A transcription factor interacts with the negative regulator RCD1 and the ACID domain of subunit 25 of the transcriptional activator mediator (Med25) to integrate stress signals for gene expression, with elusive molecular interplay. Using biophysical and structural analyses together with high-throughput screening, we revealed a bivalent binding switch in DREB2A containing a novel ACID-binding motif (ABS) and the RCD1-binding motif (RIM). The RIM is lacking in a stress-induced DREB2A splice variant with retained activity. ABS and RIM bound to separate sites on Med25-ACID, and NMR uncovered a structurally heterogeneous complex deriving from a DREB2A-ABS proline residue populating cis- and trans-isomers with remote impact on the RIM. The cis-isomer stabilized an α-helix, while the trans-isomer may introduce energetic frustration facilitating rapid exchange between activators and repressors. Thus, DREB2A uses a post-transcriptionally and post-translationally modulated switch for transcriptional regulation.

Assessment of machine‐learning predictions for the Mediator complex subunit MED25 ACID domain interactions with transactivation domains

“…7). During the revision of our manuscript, an independent biophysical analysis of AtMED25 ACID domain in complex with AtDREB2a became available [65]. Our results with AtDREB2a (252–277) are in good agreement with the NMR, ITC, MD, and AlphaFold experiments performed in this study with a longer AtDREB2a fragment (234–276).…”

“…Our results with AtDREB2a (252–277) are in good agreement with the NMR, ITC, MD, and AlphaFold experiments performed in this study with a longer AtDREB2a fragment (234–276). In particular, the authors identified two AtMED25 ACID binding motifs in AtDREB2a (234–276) [65], one of which corresponds to the one we have just highlighted in this study. However, the predicted AtMED25 binding interface appears swapped between AtDREB2a (252–277) and AtDREB2a (234–276) [65], underlining once more the importance of choosing carefully the right delimitations to optimize the accuracy of AlphaFold predictions [59,66].…”

“…In particular, the authors identified two AtMED25 ACID binding motifs in AtDREB2a (234–276) [65], one of which corresponds to the one we have just highlighted in this study. However, the predicted AtMED25 binding interface appears swapped between AtDREB2a (252–277) and AtDREB2a (234–276) [65], underlining once more the importance of choosing carefully the right delimitations to optimize the accuracy of AlphaFold predictions [59,66]. Additional AlphaFold Arabidopsis MED25 ACID domain/TADs models to guide experimental studies will help elucidate how sequence divergence between orthologs may play a role in dictating molecular recognition.…”

Assessment of machine-learning predictions for MED25 ACID domain interactions with transactivation domains

“…Our results with AtDREB2a (252-277) are in good agreement with the NMR, ITC, MD and AlphaFold experiments performed in this study with a longer AtDREB2a fragment (234-276). In particular, the authors identified two AtMED25 ACID binding motifs in AtDREB2a (234-276) (60), one of which corresponds to the one we have just highlighted in this study. Additional AlphaFold Arabidopsis MED25 ACID domain/TADs models to guide experimental studies will help elucidate how sequences divergence between orthologs may play a role in dictating molecular recognition.…”

“…The predicted shared binding interface located at the top of the β-barrel core, led us to hypothesize that hMED25 and AtMED25 ACID are structurally conserved (Figure 1) but that AtMED25-TADs interactions are specific to plants (Figure 7). During the preparation of our manuscript, an independent biophysical analysis of AtMED25 ACID domain in complex with AtDREB2a became available (60). Our results with AtDREB2a (252–277) are in good agreement with the NMR, ITC, MD and AlphaFold experiments performed in this study with a longer AtDREB2a fragment (234–276).…”