Ion binding properties of the dehydrin ERD14 are dependent upon phosphorylation

The ERD14 protein (early response to dehydration) is a member of the dehydrin family of proteins which accumulate in response to dehydration-related environmental stresses. Here we show the Arabidopsis dehydrin, ERD14, possesses ion binding properties. ERD14 is an in vitro substrate of casein kinase II; the phosphorylation resulting both in a shift in apparent molecular mass on SDS-PAGE gels and increased calcium binding activity. The phosphorylated protein bound significantly more calcium than the nonphosphorylated protein, with a dissociation constant of 120 microm and 2.86 mol of calcium bound per mol of protein. ERD14 is phosphorylated by extracts of cold-treated tissues, suggesting that the phosphorylation status of this protein might be modulated by cold-regulated kinases or phosphatases. Calcium binding properties of ERD14 purified from Arabidopsis extracts were comparable with phosphorylated Escherichia coli-expressed ERD14. Approximately 2 mol of phosphate were incorporated per mol of ERD14, indicating a minimum of two phosphorylation sites. Western blot analyses confirmed that threonine and serine are possible phosphorylation sites on ERD14. Utilizing matrix assisted laser desorption ionization-time of flight/mass spectrometry we identified five phosphorylated peptides that were present in both in vivo and in vitro phosphorylated ERD14. Our results suggest that the polyserine (S) domain is most likely the site of phosphorylation in ERD14 responsible for the activation of calcium binding.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignment of plant dehydrin early response to dehydration 10 (ERD10)

Early response to dehydration 10 protein (ERD10) is an intrinsically disordered protein from Arabidopsis thaliana. The protein is upregulated during stress however its mechanism of action at atomic level is not well understood. In the present work multidimensional NMR methodologies are used in order to facilitate the process of chemical shift assignment. The information provided here supports further NMR spectroscopy experiments aimed at elucidation of ERD10 behaviour during molecular recognition events with other proteins.     

Full backbone assignment and dynamics of the intrinsically disordered dehydrin ERD14

Dehydrins are a class of stress proteins that belong to the family of Late Embryogenesis Abundant (LEA) proteins in plants, so named because they are highly expressed in late stages of seed formation. In somatic cells, their expression is very low under normal conditions, but increases critically upon dehydration elicited by water stress, high salinity or cold. Dehydrins are thought to be intrinsically disordered proteins, which represents a challenge in understanding their structure–function relationship. Herein we present the backbone ¹H, ¹⁵N and ¹³C NMR assignment of the 185 amino acid long ERD14 (Early Response to Dehydration 14), which is a K₃S-type, typical dehydrin of A. thaliana. Secondary chemical shifts as well as NMR relaxation data show that ERD14 is fully disordered under near native conditions, with short regions of somewhat restricted motion and 5–25% helical propensity. These results suggest that ERD14 may have partially preformed elements for functional interaction with its partner(s) and set the stage for further detailed structural and functional studies of ERD14 both in vitro and in vivo.     

Phosphoproteomic analysis reveals that dehydrins ERD10 and ERD14 are phosphorylated by SNF1‐related protein kinase 2.10 in response to osmotic stress

SNF1‐related protein kinases 2 (SnRK2s) regulate the plant responses to abiotic stresses, especially water deficits. They are activated in plants subjected to osmotic stress, and some of them are additionally activated in response to enhanced concentrations of abscisic acid (ABA) in plant cells. The SnRK2s that are activated in response to ABA are key elements of ABA signalling that regulate plant acclimation to environmental stresses and ABA‐dependent development. Much less is known about the SnRK2s that are not activated by ABA, albeit several studies have shown that these kinases are also involved in response to osmotic stress. Here, we show that one of the Arabidopsis thaliana ABA‐non‐activated SnRK2s, SnRK2.10, regulates not only the response to salinity but also the plant sensitivity to dehydration. Several potential SnRK2.10 targets phosphorylated in response to stress were identified by a phosphoproteomic approach, including the dehydrins ERD10 and ERD14. Their phosphorylation by SnRK2.10 was confirmed in vitro. Our data suggest that the phosphorylation of ERD14 within the S‐segment is involved in the regulation of dehydrin subcellular localization in response to stress.     

Dehydrin ERD14 activates glutathione transferase Phi9 in Arabidopsis thaliana under osmotic stress

BackgroundFully intrinsically disordered plant dehydrin ERD14 can protect enzymes via its chaperone-like activity, but it was not formally linked with enzymes of the plant redox system yet. This is of particular interest, as the level of H₂O₂ in Arabidopsis plants increases during osmotic stress, which can be counteracted by overexpression of ERD14.MethodsThe proteomic mass-spectrometry analysis of stressed plants was performed to find the candidates affected by ERD14. With cross-linking, microscale thermophoresis, and active-site titration kinetics, the interaction and influence of ERD14 on the function of two target proteins: glutathione transferase Phi9 and catalase was examined.ResultsUnder osmotic stress, redox enzymes, specifically the glutathione transferase Phi enzymes, are upregulated. Using microscale thermophoresis, we showed that ERD14 directly interacts with GSTF9 with a K_D of ~25 μM. ERD14 activates the inactive GSTF9 molecules, protects GSTF9 from oxidation, and can also increases the activity of the enzyme. Aside from GSTF9, we found that ERD14 can also interact with catalase, an important cellular H₂O₂ scavenging enzyme, with a K_D of ~0.13 μM, and protects it from dehydration-induced loss of activity.ConclusionsWe propose that fully intrinsically disordered dehydrin ERD14 might protect and even activate redox enzymes, helping plants to survive oxidative stress under dehydration conditions.General significanceERD14 has a direct effect on the activity of redox enzymes.     

Phosphorylation regulated ion-binding is a property shared by the acidic subclass dehydrins

Dehydrins are a family of proteins that accumulate in response to abiotic stresses. Little is known about the biochemical functions of these proteins. It is known that the Arabidopsis dehydrin, ERD14, is activated by phosphorylation to bind calcium and other ions. To begin to categorize the Arabidopsis dehydrins into functional families, we determined whether representative members of the dehydrin sub families share the properties of ERD14. When phosphorylated in vitro with casein kinase II; recombinant COR47, and ERD10 (and ERD14) become activated to bind calcium. ERD14 exhibited the highest calcium-binding activity followed by ERD10 and COR47. These dehydrins, when isolated from cold-treated Arabidopsis plants were also shown to have phosphorylation-dependent, calcium-binding activity. RAB18 showed very little calcium binding activity, even though it was phosphorylated by casein kinase II. XERO2 was not phosphorylated with CKII and did not bind calcium. Competition studies suggest that other divalent cations may bind to the dehydrins COR47, ERD10, and ERD14. Utilizing matrix-assisted laser desorption ionization – time of flight mass spectroscopy (MALDI-TOF), we determined that the poly serine region located in all three calcium-binding family members (COR47, ERD10, and ERD14) is the most likely phosphorylation site responsible for the activation of calcium binding. These results are consistent with a distinct biochemical function for the acidic subclass of dehydrins (COR47, ERD10, and ERD14) as ion (calcium)-interacting proteins.     

Disordered plant LEA proteins as molecular chaperones

Plants often respond to abiotic stresses by the increased expression of LEA (late embryogenesis abundant) proteins, so called because they also accompany seed formation. Whereas the cellular function of LEA proteins in mitigating the damage caused by stress is clear, the molecular mechanisms of their action are rather enigmatic. Several models have been developed, based on their putative activities as ion sinks, stabilizers of membrane structure, buffers of hydrate water, antioxidants and/or chaperones. Due to their known structural flexibility, this latter idea has received little experimental attention thus far. Recently, however, it has been suggested that intrinsically disordered proteins (IDPs) may exert chaperone activity by an “entropy transfer” mechanism. In our subsequent study published in the May issue of Plant Physiology, we provided evidence that two group 2 LEA proteins, ERD (early response to dehydration) 10 and 14, are potent molecular chaperones. This observation may have far-reaching implications, as it may explain how LEA proteins of ill-defined structures protect plant cells during dehydration, and it may also lead to the general experimental validation of the entropy transfer model of disordered chaperones.Key words: abiotic stress, dehydration stress, stress tolerance, late embryogenesis abundant protein, chaperone, disordered protein, unstructured protein     

Chloroplast-targeted ERD1 protein declines but its mRNA increases during senescence in Arabidopsis

Arabidopsis ERD1 is a ClpC-like protein that sequence analysis suggests may interact with the chloroplast-localized ClpP protease to facilitate proteolysis. The mRNA encoded by the ERD1 gene has previously been shown to accumulate in response to senescence and to a variety of stresses and hormones. Here we show that the ERD1 protein, in contrast to the ERD1 mRNA, strongly declines in abundance with age, becoming undetectable in fully expanded leaves. Sequence analysis also suggests that ERD1 is chloroplast targeted, and we show in an in vitro system that the native protein is properly imported, processed, and present within the soluble fraction of the chloroplast, presumably the stroma. We show that ClpP protein, which is also present in the stroma, declines with age in parallel with ERD1. These results are consistent with the interaction of ERD1 and ClpP, but they suggest that it is unlikely that either plays a major role during senescence. Certain other chloroplast proteins decline with age coordinately with ERD1 and ClpP, suggesting that these declines are markers of an early age-mediated change that occurs within the chloroplast.     

Membrane topology and predicted RNA-binding function of the 'early responsive to dehydration (ERD4)' plant protein

Functional annotation of uncharacterized genes is the main focus of computational methods in the post genomic era. These tools search for similarity between proteins on the premise that those sharing sequence or structural motifs usually perform related functions, and are thus particularly useful for membrane proteins. Early responsive to dehydration (ERD) genes are rapidly induced in response to dehydration stress in a variety of plant species. In the present work we characterized function of Brassica juncea ERD4 gene using computational approaches. The ERD4 protein of unknown function possesses ubiquitous DUF221 domain (residues 312-634) and is conserved in all plant species. We suggest that the protein is localized in chloroplast membrane with at least nine transmembrane helices. We detected a globular domain of 165 amino acid residues (183-347) in plant ERD4 proteins and expect this to be posited inside the chloroplast. The structural-functional annotation of the globular domain was arrived at using fold recognition methods, which suggested in its sequence presence of two tandem RNA-recognition motif (RRM) domains each folded into βαββαβ topology. The structure based sequence alignment with the known RNA-binding proteins revealed conservation of two non-canonical ribonucleoprotein sub-motifs in both the putative RNA-recognition domains of the ERD4 protein. The function of highly conserved ERD4 protein may thus be associated with its RNA-binding ability during the stress response. This is the first functional annotation of ERD4 family of proteins that can be useful in designing experiments to unravel crucial aspects of stress tolerance mechanism.     

The binding of maize DHN1 to lipid vesicles. Gain of structure and lipid specificity

Dehydrins (DHNs; late embryogenesis abundant D-11) are a family of plant proteins induced in response to abiotic stresses such as drought, low temperature, and salinity or during the late stages of embryogenesis. Spectral and thermal properties of these proteins in purified form suggest that they are "intrinsically unstructured." However, DHNs contain at least one copy of a consensus 15-amino acid sequence, the "K segment," which resembles a class A2 amphipathic alpha-helical, lipid-binding domain found in other proteins such as apolipoproteins and alpha-synuclein. The presence of the K segment raises the question of whether DHNs bind lipids, bilayers, or phospholipid vesicles. Here, we show that maize (Zea mays) DHN DHN1 can bind to lipid vesicles that contain acidic phospholipids. We also observe that DHN1 binds more favorably to vesicles of smaller diameter than to larger vesicles, and that the association of DHN1 with vesicles results in an apparent increase of alpha-helicity of the protein. Therefore, DHNs, and presumably somewhat similar plant stress proteins in the late embryogenesis abundant and cold-regulated classes may undergo function-related conformational changes at the water/membrane interface, perhaps related to the stabilization of vesicles or other endomembrane structures under stress conditions.     

Protein-water and protein-buffer interactions in the aqueous solution of an intrinsically unstructured plant dehydrin: NMR intensity and DSC aspects

Proton NMR intensity and differential scanning calorimetry measurements were carried out on an intrinsically unstructured late embryogenesis abundant protein, ERD10, the globular BSA, and various buffer solutions to characterize water and ion binding of proteins by this novel combination of experimental approaches. By quantifying the number of hydration water molecules, the results demonstrate the interaction between the protein and NaCl and between buffer and NaCl on a microscopic level. The findings overall provide direct evidence that the intrinsically unstructured ERD10 not only has a high hydration capacity but can also bind a large amount of charged solute ions. In accord, the dehydration stress function of this protein probably results from its simultaneous action of retaining water in the drying cells and preventing an adverse increase in ionic strength, thus countering deleterious effects such as protein denaturation.     

More than just tails: intrinsic disorder in histone proteins

Many biologically active proteins are disordered as a whole, or contain long disordered regions. These intrinsically disordered proteins/regions are very common in nature, abundantly found in all organisms, where they carry out important biological functions. The functions of these proteins complement the functional repertoire of "normal" ordered proteins, and many protein functional classes are heavily dependent on intrinsic disorder. Among these disorder-centric functions are interactions with nucleic acids and protein complex assembly. In this study, we present the results of comprehensive bioinformatics analyses of the abundance and roles of intrinsic disorder in 2007 histones from 746 species. We show that all the members of the histone family are intrinsically disordered proteins. Furthermore, intrinsic disorder is not only abundant in histones, but is absolutely necessary for various histone functions, starting from heterodimerization to formation of higher order oligomers, to interactions with DNA and other proteins, and to posttranslational modifications.     

Intrinsically disordered regions of p53 family are highly diversified in evolution

Proteins of the p53 family are expressed in vertebrates and in some invertebrate species. The main function of these proteins is to control and regulate cell cycle in response to various cellular signals, and therefore to control the organism's development. The regulatory functions of the p53 family members originate mostly from their highly-conserved and well-structured DNA-binding domains. Many human diseases (including various types of cancer) are related to the missense mutations within this domain. The ordered DNA-binding domains of the p53 family members are surrounded by functionally important intrinsically disordered regions. In this study, substitution rates and propensities in different regions of p53 were analyzed. The analyses revealed that the ordered DNA-binding domain is conserved, whereas disordered regions are characterized by high sequence diversity. This diversity was reflected both in the number of substitutions and in the types of substitutions to which each amino acid was prone. These results support the existence of a positive correlation between protein intrinsic disorder and sequence divergence during the evolutionary process. This higher sequence divergence provides strong support for the existence of disordered regions in p53 in vivo for if they were structured, they would evolve at similar rates as the rest of the protein.     

固有无序蛋白质及逆境胁迫下的分子功能

固有无序蛋白质是一类在生理条件下缺乏稳定三维结构而具有正常功能,参与信号转导、转录调控、胁迫应答等多种生物学过程的蛋白质.植物中许多逆境响应蛋白是固有无序蛋白质,通过其结构无序或部分无序区域在蛋白质 蛋白质、蛋白质 膜脂、蛋白质 核酸的互作中发挥重要作用.本文主要对固有无序蛋白质的类别、氨基酸组成和结构特点以及在逆境胁迫下其稳定细胞膜、保护核酸和蛋白质、调控基因表达等分子功能进行综述,以拓展对逆境胁迫下蛋白质作用分子机制的认识.     

Structured proteins and proteins with intrinsic disorder

Until recently, the point of view that the unique tertiary structure is necessary for protein function has prevailed. However, recent data have demonstrated that many cell proteins do not possess such structure in isolation, although displaying a distinct function under physiological conditions. These proteins were named the naturally, or intrinsically, disordered proteins. The fraction of intrinsically disordered regions in such proteins may vary from several amino acid residues to a completely unordered sequence of several tens or even several hundreds of residues. The main distinction of these proteins from structured (globular) proteins is that they have no unique tertiary structure in isolation and acquire it only upon interaction with their partners. The conformation of these proteins in a complex is determined not only by their own amino acid sequence (as is typical of structured, or globular, proteins) but also by the interacting partner. This review discusses the structure-function relationships in structured and intrinsically disordered proteins. The intricateness of this problem and the possible ways to solve it are illustrated by the example of the EF1A elongation factor family.     

Influence of drying on the secondary structure of intrinsically disordered and globular proteins

Circular dichroism (CD) spectroscopy of five Arabidopsis late embryogenesis abundant (LEA) proteins constituting the plant specific families LEA_5 and LEA_6 showed that they are intrinsically disordered in solution and partially fold during drying. Structural predictions were comparable to these results for hydrated LEA_6, but not for LEA_5 proteins. FTIR spectroscopy showed that verbascose, but not sucrose, strongly affected the structure of the dry proteins. The four investigated globular proteins were only mildly affected by drying in the absence, but strongly in the presence of sugars. These data highlight the larger structural flexibility of disordered compared to globular proteins and the impact of sugars on the structure of both disordered and globular proteins during drying.