Intrinsic Disorder in Plant Transcription Factor Systems: Functional Implications

Abstract

Eukaryotic cells are complex biological systems that depend on highly connected molecular interaction networks with intrinsically disordered proteins as essential components. Through specific examples, we relate the conformational ensemble nature of intrinsic disorder (ID) in transcription factors to functions in plants. Transcription factors contain large regulatory ID-regions with numerous orphan sequence motifs, representing potential important interaction sites. ID-regions may affect DNA-binding through electrostatic interactions or allosterically as for the bZIP transcription factors, in which the DNA-binding domains also populate ensembles of dynamic transient structures. The flexibility of ID is well-suited for interaction networks requiring efficient molecular adjustments. For example, Radical Induced Cell Death1 depends on ID in transcription factors for its numerous, structurally heterogeneous interactions, and the JAZ:MYC:MED15 regulatory unit depends on protein dynamics, including binding-associated unfolding, for regulation of jasmonate-signaling. Flexibility makes ID-regions excellent targets of posttranslational modifications. For example, the extent of phosphorylation of the NAC transcription factor SOG1 regulates target gene expression and the DNA-damage response, and phosphorylation of the AP2/ERF transcription factor DREB2A acts as a switch enabling heat-regulated degradation. ID-related phase separation is emerging as being important to transcriptional regulation with condensates functioning in storage and inactivation of transcription factors. The applicative potential of ID-regions is apparent, as removal of an ID-region of the AP2/ERF transcription factor WRI1 affects its stability and consequently oil biosynthesis. The highlighted examples show that ID plays essential functional roles in plant biology and has a promising potential in engineering.

Keywords: ID; activation domain; coupled folding and binding; electrostatic interactions; interactome; phase separation; posttranslational modification; sequence motif.

Figure 1

Schematic representations of the domain structures, post translational moodification sites, order-disorder profiles and MoRF profiles of plant TFs with known ID-based functions. The TF shown are from the following TF families: (A) NAC; (B) AR2/ERF; (C) bHLH; (D) MYB; (E) bZIP; (F) TCP; (G) ARF. ID was predicted using IUPred2A (blue line) and PONDR-FIT (VSL2) (red line), and MoRFs were predicted using MoRFpred (black line). The disorder threshold is 0.5 with disorder assigned to values larger than or equal to 0.5.

Figure 2

TF-ID in regulatory interactions (A) ID-based interactomes and mechanisms of interactions. (A) RCD1 and DREB2A interactomes curated from the STRING database, with medium confidence score (0.400) and including only experimental interactions. (B) Model of RCD1:TF interactions involving structural heterogeneity in the complexes. Several TFs, including DREB2A and ANAC013, may compete for binding to RCD1. Some TFs, e.g., DREB2A, undergo coupled folding and binding upon association with RCD1-RST, whereas other, e.g., ANAC013, may keep disorder in the complexes. RCD1 negatively regulates its TF interaction partners. The structure of the RCD1-RST domain (PDB 5OAO) is visualized using PyMOL. (C) Mechanisms of coupled folding and binding: conformational selection (CS) and induced fit (IF). In CS, a conformation from the TF ensemble is selected for binding by the partner protein. In IF, the TF recognizes its partner in a disordered state that folds after binding to the partner protein. (D) MYC3 in free (PDB 4RRU) and JAZ9-bound (PDB 4YWC) states. The Jas-motif of JAZ9 (purple) binds the N-terminal domain (grey) of MYC3, displacing the α1 helix (green) and rearranging the JID helix (cyan) of MYC3. The MYC3:JAZ9 complex cannot bind MED25 and is therefore not transcriptionally active. Upon release of JA hormone, JAZ9 is released from MYC3 and degraded through the ubiquitin-proteasome pathway enabling MED25:MYC3 complex formation. (E) The MYB–MIM:bHLH interaction is context dependent. The MIM SLiM, present in sub-group 12 MYB TFs as MYB29, only interacts with the bHLH TF MYC4 when present in its natural IDR context and not when inserted in the IDR context of e.g., MYB75.

Figure 3

Regulation of biological function by PTMs of plant TF IDR as in the case of SOG1 phosphorylation in the DDR. Upon DNA damage, ATM (kinase) is activated and phosphorylates five SQ motifs in SOG1. SOG1 regulates the expression of genes associated with DDR, cell cycle regulation, and programmed cell death. The mechanisms of this activation of SOG1 remains unknown, but phosphorylation of the SOG1 IDR may affect the exposure of the DBD through long-range electrostatic interactions between the IDR and the DBD.

Figure 4

Phase separation in transcriptional regulation in plants. (A) The TF VRN1 undergoes phase separation in the presence of DNA to form organelles repressing gene expression. (B) ARF TFs promote cellular growth and differentiation through DNA-binding in the nucleus. In the stationary phase cells, the TFs are inactive in cytoplasmic condensates. (C) ELF3 regulation is temperature dependent: at 22 °C the protein is free and binds DNA to repress transcription, at 27 °C it undergoes protein-protein demixing to promote the genes expression.

Figure 5

Effects of IDRs on DNA-binding. For ANAC019, a histidine switch affects DNA-binding. A “perfect” DBD (cylinder) dimer is formed, when His135 is deprotonated, which allows to stabilize interactions between residues of the two subunits of ANAC019, allowing higher affinities in DNA-binding. When His135 is protonated, a salt-bridge disrupts most of these interactions. Removal of the ANAC019 IDR abolishes the pH-dependence of DNA-binding, suggesting that the negatively charged disordered AD tunes the DNA-binding affinity of the DBD.