αα-hub coregulator structure and flexibility determine transcription factor binding and selection in regulatory interactomes.

Formation of transcription factor (TF)-coregulator complexes is a key step in transcriptional regulation, with coregulators having essential functions as hub nodes in molecular networks. How specificity and selectivity are maintained in these nodes remain open questions. In this work, we addressed specificity in transcriptional networks using complexes formed between TFs and αα-hubs, which are defined by a common αα-hairpin secondary structure motif, as a model. Using NMR spectroscopy and binding thermodynamics, we analyzed the structure, dynamics, stability, and ligand-binding properties of the Arabidopsis thaliana RST domains from TAF4 and known binding partner RCD1, and the TAFH domain from human TAF4, allowing comparison across species, functions, and architectural contexts. While these αα-hubs shared the αα-hairpin motif, they differed in length and orientation of accessory helices as well as in their thermodynamic profiles of ligand binding. Whereas biologically relevant RCD1-ligand pairs displayed high affinity driven by enthalpy, TAF4-ligand interactions were entropy driven and exhibited less binding-induced structuring. We in addition identified a thermal unfolding state with a structured core for all three domains, although the temperature sensitivity differed. Thermal stability studies suggested that initial unfolding of the RCD1-RST domain localized around helix 1, lending this region structural malleability, while effects in TAF4-RST were more stochastic, suggesting variability in structural adaptability upon binding. Collectively, our results support a model in which hub structure, flexibility, and binding thermodynamics contribute to αα-hub-TF binding specificity, a finding of general relevance to the understanding of coregulator-ligand interactions and interactome sizes.

αα-Hub domains and intrinsically disordered proteins: A decisive combo.

Hub proteins are central nodes in protein-protein interaction networks with critical importance to all living organisms. Recently, a new group of folded hub domains, the αα-hubs, was defined based on a shared αα-hairpin supersecondary structural foundation. The members PAH, RST, TAFH, NCBD, and HHD are found in large proteins such as Sin3, RCD1, TAF4, CBP, and harmonin, which organize disordered transcriptional regulators and membrane scaffolds in interactomes of importance to human diseases and plant quality. In this review, studies of structures, functions, and complexes across the αα-hubs are described and compared to provide a unified description of the group. This analysis expands the associated molecular concepts of "one domain-one binding site", motif-based ligand binding, and coupled folding and binding of intrinsically disordered ligands to additional concepts of importance to signal fidelity. These include context, motif reversibility, multivalency, complex heterogeneity, synergistic αα-hub:ligand folding, accessory binding sites, and supramodules. We propose that these multifaceted protein-protein interaction properties are made possible by the characteristics of the αα-hub fold, including supersite properties, dynamics, variable topologies, accessory helices, and malleability and abetted by adaptability of the disordered ligands. Critically, these features provide additional filters for specificity. With the presentations of new concepts, this review opens for new research questions addressing properties across the group, which are driven from concepts discovered in studies of the individual members. Combined, the members of the αα-hubs are ideal models for deconvoluting signal fidelity maintained by folded hubs and their interactions with intrinsically disordered ligands.

Germin like protein genes exhibit modular expression during salt and drought stress in elite rice cultivars.

BACKGROUND: Germin-like proteins (GLPs) are ubiquitous plant proteins, which play significant role in plant responses against various abiotic stresses. However, the potential functions of GLPs in rice (Oryza sativa) against salt and drought stress are still unclear. METHODS AND RESULTS: In this study, transcriptional variation of eight OsGLP genes (OsGLP3-6, OsGLP4-1, OsGLP8-4, OsGLP8-7, OsGLP8-10, OsGLP8-11 and OsGLP8-12) was analyzed in leaves and roots of two economically important Indica rice cultivars, KS282 and Super Basmati, under salt and drought stress at early seedling stage. The relative expression analysis from qRT-PCR indicated the highest increase in expression of OsGLP3-6 in leaves and roots of both rice varieties with a significantly higher expression in KS282. Moreover, relative change in expression of OsGLP8-7, OsGLP8-10 and OsGLP8-11 under salt stress and OsGLP8-7 under drought stress was also commonly higher in leaves and roots of KS282 as compared to Super Basmati. Whereas, OsGLP3-7 and OsGLP8-12 after salt stress and OsGLP8-4 and OsGLP8-12 after drought stress were observed with higher relative expression in roots of Super Basmati than KS282. Importantly, the OsGLP3-6 and OsGLP4-1 from chromosome 3 and 4 respectively showed higher expression in leaves whereas most of the OsGLP genes from chromosome 8 exhibited higher expression in roots. CONCLUSION: Overall, as a result of this comparative analysis, OsGLP genes showed both general and specific expression profiles depending upon a specific rice variety, stress condition as well as tissue type. These results will increase our understanding of role of OsGLP genes in rice crop and provide useful information for the further in-depth research on their regulatory mechanisms in response to these stress conditions.

Disorder in a two-domain neuronal Ca2+-binding protein regulates domain stability and dynamics using ligand mimicry.

Understanding the interplay between sequence, structure and function of proteins has been complicated in recent years by the discovery of intrinsically disordered proteins (IDPs), which perform biological functions in the absence of a well-defined three-dimensional fold. Disordered protein sequences account for roughly 30% of the human proteome and in many proteins, disordered and ordered domains coexist. However, few studies have assessed how either feature affects the properties of the other. In this study, we examine the role of a disordered tail in the overall properties of the two-domain, calcium-sensing protein neuronal calcium sensor 1 (NCS-1). We show that loss of just six of the 190 residues at the flexible C-terminus is sufficient to severely affect stability, dynamics, and folding behavior of both ordered domains. We identify specific hydrophobic contacts mediated by the disordered tail that may be responsible for stabilizing the distal N-terminal domain. Moreover, sequence analyses indicate the presence of an LSL-motif in the tail that acts as a mimic of native ligands critical to the observed order-disorder communication. Removing the disordered tail leads to a shorter life-time of the ligand-bound complex likely originating from the observed destabilization. This close relationship between order and disorder may have important implications for how investigations into mixed systems are designed and opens up a novel avenue of drug targeting exploiting this type of behavior.

Quantification of Conformational Entropy Unravels Effect of Disordered Flanking Region in Coupled Folding and Binding.

Intrinsic disorder (ID) constitutes a new dimension to the protein structure-function relationship. The ability to undergo conformational changes upon binding is a key property of intrinsically disordered proteins and remains challenging to study using conventional methods. A 1994 paper by R. S. Spolar and M. T. Record presented a thermodynamic approach for estimating changes in conformational entropy based on heat capacity changes, allowing quantification of residues folding upon binding. Here, we adapt the method for studies of intrinsically disordered proteins. We integrate additional data to provide a broader experimental foundation for the underlying relations and, based on >500 protein-protein complexes involving disordered proteins, reassess a key relation between polar and nonpolar surface area changes, previously determined using globular protein folding. We demonstrate the improved suitability of the adapted method to studies of the folded αα-hub domain RST from radical-induced cell death 1, whose interactome is characterized by ID. From extensive thermodynamic data, quantifying the conformational entropy changes upon binding, and comparison to the NMR structure, the adapted method improves accuracy for ID-based studies. Furthermore, we apply the method, in conjunction with NMR, to reveal hitherto undetected effects of interaction-motif context. Thus, inclusion of the disordered context of the DREB2A RST-binding motif induces structuring of the binding motif, resulting in major enthalpy-entropy compensation in the interaction interface. This study, also evaluating additional interactions, demonstrates the strength of the ID-adapted Spolar-Record thermodynamic approach for dissection of structural features of ID-based interactions, easily overlooked in traditional studies, and for translation of these into mechanistic knowledge.

Connecting the αα-hubs: same fold, disordered ligands, new functions.

BACKGROUND: Signal fidelity depends on protein-protein interaction-'hubs' integrating cues from large interactomes. Recently, and based on a common secondary structure motif, the αα-hubs were defined, which are small α-helical domains of large, modular proteins binding intrinsically disordered transcriptional regulators. METHODS: Comparative structural biology. RESULTS: We assign the harmonin-homology-domain (HHD, also named the harmonin N-terminal domain, NTD) present in large proteins such as harmonin, whirlin, cerebral cavernous malformation 2, and regulator of telomere elongation 1 to the αα-hubs. The new member of the αα-hubs expands functionality to include scaffolding of supra-modular complexes mediating sensory perception, neurovascular integrity and telomere regulation, and reveal novel features of the αα-hubs. As a common trait, the αα-hubs bind intrinsically disordered ligands of similar properties integrating similar cellular cues, but without cross-talk. CONCLUSION: The inclusion of the HHD in the αα-hubs has uncovered new features, exemplifying the utility of identifying groups of hub domains, whereby discoveries in one member may cross-fertilize discoveries in others. These features make the αα-hubs unique models for decomposing signal specificity and fidelity. Using these as models, together with other suitable hub domain, we may advance the functional understanding of hub proteins and their role in cellular communication and signaling, as well as the role of intrinsically disordered proteins in signaling networks. Video Abstract.

A single class of ARF GTPase activated by several pathway-specific ARF-GEFs regulates essential membrane traffic in Arabidopsis.

In eukaryotes, GTP-bound ARF GTPases promote intracellular membrane traffic by mediating the recruitment of coat proteins, which in turn sort cargo proteins into the forming membrane vesicles. Mammals employ several classes of ARF GTPases which are activated by different ARF guanine-nucleotide exchange factors (ARF-GEFs). In contrast, flowering plants only encode evolutionarily conserved ARF1 GTPases (class I) but not the other classes II and III known from mammals, as suggested by phylogenetic analysis of ARF family members across the five major clades of eukaryotes. Instead, flowering plants express plant-specific putative ARF GTPases such as ARFA and ARFB, in addition to evolutionarily conserved ARF-LIKE (ARL) proteins. Here we show that all eight ARF-GEFs of Arabidopsis interact with the same ARF1 GTPase, whereas only a subset of post-Golgi ARF-GEFs also interacts with ARFA, as assayed by immunoprecipitation. Both ARF1 and ARFA were detected at the Golgi stacks and the trans-Golgi network (TGN) by both live-imaging with the confocal microscope and nano-gold labeling followed by EM analysis. ARFB representing another plant-specific putative ARF GTPase was detected at both the plasma membrane and the TGN. The activation-impaired form (T31N) of ARF1, but neither ARFA nor ARFB, interfered with development, although ARFA-T31N interfered, like ARF1-T31N, with the GDP-GTP exchange. Mutant plants lacking both ARFA and ARFB transcripts were viable, suggesting that ARF1 is sufficient for all essential trafficking pathways under laboratory conditions. Detailed imaging of molecular markers revealed that ARF1 mediated all known trafficking pathways whereas ARFA was not essential to any major pathway. In contrast, the hydrolysis-impaired form (Q71L) of both ARF1 and ARFA, but not ARFB, had deleterious effects on development and various trafficking pathways. However, the deleterious effects of ARFA-Q71L were abolished by ARFA-T31N inhibiting cognate ARF-GEFs, both in cis (ARFA-T31N,Q71L) and in trans (ARFA-T31N + ARFA-Q71L), suggesting indirect effects of ARFA-Q71L on ARF1-mediated trafficking. The deleterious effects of ARFA-Q71L were also suppressed by strong over-expression of ARF1, which was consistent with a subset of BIG1-4 ARF-GEFs interacting with both ARF1 and ARFA. Indeed, the SEC7 domain of BIG5 activated both ARF1 and ARFA whereas the SEC7 domain of BIG3 only activated ARF1. Furthermore, ARFA-T31N impaired root growth if ARF1-specific BIG3 was knocked out and only ARF1- and ARFA-activating BIG4 was functional. Activated ARF1 recruits different coat proteins to different endomembrane compartments, depending on its activation by different ARF-GEFs. Unlike ARF GTPases, ARF-GEFs not only localize at distinct compartments but also regulate specific trafficking pathways, suggesting that ARF-GEFs might play specific roles in traffic regulation beyond the activation of ARF1 by GDP-GTP exchange.

Intrinsic Disorder in Plant Transcription Factor Systems: Functional Implications.

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.

Structures and Short Linear Motif of Disordered Transcription Factor Regions Provide Clues to the Interactome of the Cellular Hub Protein Radical-induced Cell Death1.

Intrinsically disordered protein regions (IDRs) lack a well defined three-dimensional structure but often facilitate key protein functions. Some interactions between IDRs and folded protein domains rely on short linear motifs (SLiMs). These motifs are challenging to identify, but once found they can point to larger networks of interactions, such as with proteins that serve as hubs for essential cellular functions. The stress-associated plant protein radical-induced cell death1 (RCD1) is one such hub, interacting with many transcription factors via their flexible IDRs. To identify the SLiM bound by RCD1, we analyzed the IDRs in three protein partners, DREB2A (dehydration-responsive element-binding protein 2A), ANAC013, and ANAC046, considering parameters such as disorder, context, charges, and pI. Using a combined bioinformatics and experimental approach, we have identified the bipartite RCD1-binding SLiM as (DE)X(1,2)(YF)X(1,4)(DE)L, with essential contributions from conserved aromatic, acidic, and leucine residues. Detailed thermodynamic analysis revealed both favorable and unfavorable contributions from the IDRs surrounding the SLiM to the interactions with RCD1, and the SLiM affinities ranged from low nanomolar to 50 times higher Kd values. Specifically, although the SLiM was surrounded by IDRs, individual intrinsic α-helix propensities varied as shown by CD spectroscopy. NMR spectroscopy further demonstrated that DREB2A underwent coupled folding and binding with α-helix formation upon interaction with RCD1, whereas peptides from ANAC013 and ANAC046 formed different structures or were fuzzy in the complexes. These findings allow us to present a model of the stress-associated RCD1-transcription factor interactome and to contribute to the emerging understanding of the interactions between folded hubs and their intrinsically disordered partners.

Eukaryotic transcription factors: paradigms of protein intrinsic disorder.

Gene-specific transcription factors (TFs) are key regulatory components of signaling pathways, controlling, for example, cell growth, development, and stress responses. Their biological functions are determined by their molecular structures, as exemplified by their structured DNA-binding domains targeting specific cis-acting elements in genes, and by the significant lack of fixed tertiary structure in their extensive intrinsically disordered regions. Recent research in protein intrinsic disorder (ID) has changed our understanding of transcriptional activation domains from 'negative noodles' to ID regions with function-related, short sequence motifs and molecular recognition features with structural propensities. This review focuses on molecular aspects of TFs, which represent paradigms of ID-related features. Through specific examples, we review how the ID-associated flexibility of TFs enables them to participate in large interactomes, how they use only a few hydrophobic residues, short sequence motifs, prestructured motifs, and coupled folding and binding for their interactions with co-activators, and how their accessibility to post-translational modification affects their interactions. It is furthermore emphasized how classic biochemical concepts like allostery, conformational selection, induced fit, and feedback regulation are undergoing a revival with the appreciation of ID. The review also describes the most recent advances based on computational simulations of ID-based interaction mechanisms and structural analysis of ID in the context of full-length TFs and suggests future directions for research in TF ID.

Barley plants over-expressing the NAC transcription factor gene HvNAC005 show stunting and delay in development combined with early senescence.

The plant-specific NAC transcription factors have attracted particular attention because of their involvement in stress responses, senescence, and nutrient remobilization. The HvNAC005 gene of barley encodes a protein belonging to subgroup NAC-a6 of the NAC family. This study shows that HvNAC005 is associated with developmental senescence. It was significantly up-regulated following ABA treatment, supported by ABA-responsive elements in its promoter, but it was not up-regulated during dark-induced senescence. The C-termini of proteins closely related to HvNAC005 showed overall high divergence but also contained conserved short motifs. A serine- and leucine-containing central motif was essential for transcriptional activity of the HvNAC005 C-terminus in yeast. Over-expression of HvNAC005 in barley resulted in a strong phenotype with delayed development combined with precocious senescence. The over-expressing plants showed up-regulation of genes involved with secondary metabolism, hormone metabolism, stress, signalling, development, and transport. Up-regulation of senescence markers and hormone metabolism and signalling genes supports a role of HvNAC005 in the cross field of different hormone and signalling pathways. Binding of HvNAC005 to promoter sequences of putative target genes containing the T[G/A]CGT core motif was shown by direct protein-DNA interactions of HvNAC005 with promoters for two of the up-regulated genes. In conclusion, HvNAC005 was shown to be a strong positive regulator of senescence and so is an obvious target for the fine-tuning of gene expression in future attempts to improve nutrient remobilization related to the senescence process in barley.

Protein intrinsic disorder in Arabidopsis NAC transcription factors: transcriptional activation by ANAC013 and ANAC046 and their interactions with RCD1.

Protein ID (intrinsic disorder) plays a significant, yet relatively unexplored role in transcription factors (TFs). In the present paper, analysis of the transcription regulatory domains (TRDs) of six phylogenetically representative, plant-specific NAC [no apical meristem, ATAF (Arabidopsis transcription activation factor), cup-shaped cotyledon] TFs shows that the domains are present in similar average pre-molten or molten globule-like states, but have different patterns of order/disorder and MoRFs (molecular recognition features). ANAC046 (Arabidopsis NAC 046) was selected for further studies because of its simple MoRF pattern and its ability to interact with RCD1 (radical-induced cell death 1). Experiments in yeast and thermodynamic characterization suggest that its single MoRF region is sufficient for both transcriptional activation and interaction with RCD1. The remainder of the large regulatory domain is unlikely to contribute to the interaction, since the domain and truncations thereof have similar affinities for RCD1, which are also similar for ANAC013-RCD1 interactions. However, different enthalpic and entropic contributions to binding were revealed for ANAC046 and ANAC013, suggestive of differences in binding mechanisms. Although substitution of both hydrophobic and acidic residues of the ANAC046 MoRF region abolished binding, substitution of other residues, even with α-helix-breaking proline, was less disruptive. Together, the biophysical analyses suggest that RCD1-ANAC046 complex formation does not involve folding-upon-binding, but rather fuzziness or an unknown structure in ANAC046. We suggest that the ANAC046 regulatory domain functions as an entropic chain with a terminal hot spot interacting with RCD1. RCD1, a cellular hub, may be able to interact with many different TFs by exploiting their ID-based flexibility, as demonstrated for its interactions with ANAC046 and ANAC013.

A DNA-binding-site landscape and regulatory network analysis for NAC transcription factors in Arabidopsis thaliana.

Target gene identification for transcription factors is a prerequisite for the systems wide understanding of organismal behaviour. NAM-ATAF1/2-CUC2 (NAC) transcription factors are amongst the largest transcription factor families in plants, yet limited data exist from unbiased approaches to resolve the DNA-binding preferences of individual members. Here, we present a TF-target gene identification workflow based on the integration of novel protein binding microarray data with gene expression and multi-species promoter sequence conservation to identify the DNA-binding specificities and the gene regulatory networks of 12 NAC transcription factors. Our data offer specific single-base resolution fingerprints for most TFs studied and indicate that NAC DNA-binding specificities might be predicted from their DNA-binding domain's sequence. The developed methodology, including the application of complementary functional genomics filters, makes it possible to translate, for each TF, protein binding microarray data into a set of high-quality target genes. With this approach, we confirm NAC target genes reported from independent in vivo analyses. We emphasize that candidate target gene sets together with the workflow associated with functional modules offer a strong resource to unravel the regulatory potential of NAC genes and that this workflow could be used to study other families of transcription factors.

NAC transcription factor gene regulatory and protein-protein interaction networks in plant stress responses and senescence.

Plant-specific NAM/ATAF/CUC (NAC) transcription factors (TFs) have recently received considerable attention due to their significant roles in plant development and stress signaling. Here, we summarize progress in understanding NAC TFs in stress responses and senescence. We focus on interactions between the DNA-binding NAC domain and target genes, and between the large, mostly disordered transcription regulatory domain of NAC TFs and protein interaction partners. Recent studies have identified both up-stream regulators of NAC genes and down-stream NAC target genes, outlining regulatory networks associated with NAC-protein interactions. This connects molecular interactions and signal pathway intersections with biological functions with promising use in agriculture. © 2014 IUBMB Life, 66(3):156-166, 2014.

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

The Arabidopsis thaliana DREB2A transcription factor interacts with the negative regulator RCD1 and the ACID domain of subunit 25 of the transcriptional co-regulator mediator (Med25) to integrate stress signals for gene expression, with elusive molecular interplay. Using biophysical and structural analyses together with high-throughput screening, we reveal a bivalent binding switch in DREB2A containing an ACID-binding motif (ABS) and the known RCD1-binding motif (RIM). The RIM is lacking in a stress-induced DREB2A splice variant with retained transcriptional activity. ABS and RIM bind to separate sites on Med25-ACID, and NMR analyses show 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 stabilizes 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.

Introduction of a tryptophan side chain into subsite +1 enhances transglycosylation activity of a GH-18 chitinase from Arabidopsis thaliana, AtChiC.

A tryptophan side chain was introduced into subsite +1 of family GH-18 (class V) chitinases from Nicotiana tabacum and Arabidopsis thaliana (NtChiV and AtChiC, respectively) by the mutation of a glycine residue to tryptophan (G74W-NtChiV and G75W-AtChiC). The specific activity toward glycol chitin of the two mutant enzymes was 70-71% of that of the wild type. Using chitin oligosaccharides, (GlcNAc)(n) (n = 4, 5 and 6), as the substrates, we found the transglycosylation reaction to be significantly enhanced in G74W-NtChiV and G75W-AtChiC when compared with the corresponding wild-type enzymes. The introduced tryptophan side chain might protect the oxazolinium ion intermediate from attack by a nucleophilic water molecule. The enhancement of transglycosylation activity was much more distinct in G75W-AtChiC than in G74W-NtChiV. Nuclear magnetic resonance titration experiments using the inactive double mutants, E115Q/G74W-NtChiV and E116Q/G75W-AtChiC revealed that the association constant of (GlcNAc)(5) was considerably larger for the latter. Amino acid substitutions at the acceptor binding site might have resulted in the larger association constant for G75W-AtChiC, giving rise to the higher transglycosylation activity of G75W-AtChiC.

DNA binding by the plant-specific NAC transcription factors in crystal and solution: a firm link to WRKY and GCM transcription factors.

NAC (NAM/ATAF/CUC) plant transcription factors regulate essential processes in development, stress responses and nutrient distribution in important crop and model plants (rice, Populus, Arabidopsis), which makes them highly relevant in the context of crop optimization and bioenergy production. The structure of the DNA-binding NAC domain of ANAC019 has previously been determined by X-ray crystallography, revealing a dimeric and predominantly ß-fold structure, but the mode of binding to cognate DNA has remained elusive. In the present study, information from low resolution X-ray structures and small angle X-ray scattering on complexes with oligonucleotides, mutagenesis and (DNase I and uranyl photo-) footprinting, is combined to form a structural view of DNA-binding, and for the first time provide experimental evidence for the speculated relationship between plant-specific NAC proteins, WRKY transcription factors and the mammalian GCM (Glial cell missing) transcription factors, which all use a ß-strand motif for DNA-binding. The structure shows that the NAC domain inserts the edge of its core ß-sheet into the major groove, while leaving the DNA largely undistorted. The structure of the NAC-DNA complex and a new crystal form of the unbound NAC also indicate limited flexibility of the NAC dimer arrangement, which could be important in recognizing suboptimal binding sites.

Structure, function and networks of transcription factors involved in abiotic stress responses.

Transcription factors (TFs) are master regulators of abiotic stress responses in plants. This review focuses on TFs from seven major TF families, known to play functional roles in response to abiotic stresses, including drought, high salinity, high osmolarity, temperature extremes and the phytohormone ABA. Although ectopic expression of several TFs has improved abiotic stress tolerance in plants, fine-tuning of TF expression and protein levels remains a challenge to avoid crop yield loss. To further our understanding of TFs in abiotic stress responses, emerging gene regulatory networks based on TFs and their direct targets genes are presented. These revealed components shared between ABA-dependent and independent signaling as well as abiotic and biotic stress signaling. Protein structure analysis suggested that TFs hubs of large interactomes have extended regions with protein intrinsic disorder (ID), referring to their lack of fixed tertiary structures. ID is now an emerging topic in plant science. Furthermore, the importance of the ubiquitin-proteasome protein degradation systems and modification by sumoylation is also apparent from the interactomes. Therefore; TF interaction partners such as E3 ubiquitin ligases and TF regions with ID represent future targets for engineering improved abiotic stress tolerance in crops.

Conformational entropy in molecular recognition of intrinsically disordered proteins.

Broad conformational ensembles make intrinsically disordered proteins or regions entropically intriguing. Although methodologically challenging and understudied, emerging studies into their changes in conformational entropy (ΔS°conf) upon complex formation have provided both quantitative and qualitative insight. Recent work based on thermodynamics from isothermal titration calorimetry and NMR spectroscopy uncovers an expanded repertoire of regulatory mechanisms, where ΔS°conf plays roles in partner selection, state behavior, functional buffering, allosteric regulation, and drug design. We highlight these mechanisms to display the large entropic reservoir of IDPs for the regulation of molecular communication. We call upon the field to make efforts to contribute to this insight as more studies are needed for forwarding mechanistic decoding of intrinsically disordered proteins and their complexes.

Evolutionary fine-tuning of residual helix structure in disordered proteins manifests in complex structure and lifetime.

Transcription depends on complex networks, where folded hub proteins interact with intrinsically disordered transcription factors undergoing coupled folding and binding. For this, local residual structure, a prototypical feature of intrinsic disorder, is key. Here, we dissect the unexplored functional potential of residual structure by comparing structure, kinetics, and thermodynamics within the model system constituted of the DREB2A transcription factor interacting with the αα-hub RCD1-RST. To maintain biological relevance, we developed an orthogonal evolutionary approach for the design of variants with varying amounts of structure. Biophysical analysis revealed a correlation between the amount of residual helical structure and binding affinity, manifested in altered complex lifetime due to changed dissociation rate constants. It also showed a correlation between helical structure in free and bound DREB2A variants. Overall, this study demonstrated how evolution can balance and fine-tune residual structure to regulate complexes in coupled folding and binding, potentially affecting transcription factor competition.