Abstract
Radical-Induced Cell Death1 (RCD1) functions as a cellular hub interacting with intrinsically disordered transcription factor regions, which lack a well-defined three-dimensional structure, to regulate plant stress. Here, we address the molecular evolution of the RCD1-interactome. Using bioinformatics, its history was traced back more than 480 million years to the emergence of land plants with the RCD1-binding short linear motif (SLiM) identified from mosses to flowering plants. SLiM variants were biophysically verified to be functional and to depend on the same RCD1 residues as the DREB2A transcription factor. Based on this, numerous additional members may be assigned to the RCD1-interactome. Conservation was further strengthened by similar intrinsic disorder profiles of the transcription factor homologs. The unique structural plasticity of the RCD1-interactome, with RCD1-binding induced α-helix formation in DREB2A, but not detectable in ANAC046 or ANAC013, is apparently conserved. Thermodynamic analysis also indicated conservation with interchangeability between Arabidopsis and soybean RCD1 and DREB2A, although with fine-tuned co-evolved binding interfaces. Interruption of conservation was observed, as moss DREB2 lacked the SLiM, likely reflecting differences in plant stress responses. This whole-interactome study uncovers principles of the evolution of SLiM:hub-interactions, such as conservation of α-helix propensities, which may be paradigmatic for disorder-based interactomes in eukaryotes.
Highlights
Most functional proteins fold into well-defined structures
Downregulation of AtRCD1 or loss of the Radical-Induced Cell Death1 (RCD1)-interacting site of DREB2A is required for proper DREB2A function under stress conditions[32], and inactivation of RCD1 resulted in increased expression of Arabidopsis No-apical-meristem, Arabidopsis transcription activation factor, Cup-shaped cotyledon (ANAC)[013] and ANAC017-regulated genes from the mitochondrial dysfunction stimulon[33]
This study demonstrates that the RBS of the intrinsic disorder (ID)-based RCD1:transcription factors (TFs) interactome arose in the land plant lineage 480 mill. years ago (Fig. 2A) and is evolutionary highly conserved
Summary
Most functional proteins fold into well-defined structures. 30–50% of eukaryotic proteins contain large regions of intrinsic disorder (ID)[1,2] allowing function without well-defined folds[3,4,5,6,7,8]. The plant hub-protein Radical-Induced Cell Death 1 (RCD1) represents a suitable model for studies of interactions between folded hub domains and SLiMs in disordered targets, and allows translation from in vitro to the organismal level[23]. AtRCD1 plays roles in hormone signaling, responses to reactive oxygen species and other abiotic stress factors, immunity and development[26,27,28,29] It interacts with numerous TFs (Fig. 1C)[26,30], including Dehydration-Responsive Element-Binding protein (DREB) 2 A, implicated in abiotic stress responses[31]. The evolution of the RCD1:TF interactome was analyzed This revealed evolutionary conservation of RCD1, the RBS and the TF order-disorder patterns in land plants over a period of 480 million years. Our whole-interactome approach, spurred by ID profiles and motif based interactions in a molecularly intriguing interactome, is readily transferrable to other ID-based interactomes
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