Assessing the dynamics and macromolecular interactions of the intrinsically disordered protein YY1.

YY1 is a ubiquitously expressed, intrinsically disordered transcription factor involved in neural development. The oligomeric state of YY1 varies depending on the environment. These structural changes may alter its DNA binding ability and hence its transcriptional activity. Just as YY1's oligomeric state can impact its role in transcription, so does its interaction with other proteins such as FOXP2. The aim of this work is to study the structure and dynamics of YY1 so as to determine the influence of oligomerisation and associations with FOXP2 on its DNA binding mechanism. The results confirm that YY1 is primarily a disordered protein, but it does consist of certain specific structured regions. We observed that YY1 quaternary structure is a heterogenous mixture of oligomers, the overall size of which is dependent on ionic strength. Both YY1 oligomerisation and its dynamic behaviour are further subject to changes upon DNA binding, whereby increases in DNA concentration result in a decrease in the size of YY1 oligomers. YY1 and the FOXP2 forkhead domain were found to interact with each other both in isolation and in the presence of YY1-specific DNA. The heterogeneous, dynamic multimerisation of YY1 identified in this work is, therefore likely to be important for its ability to make heterologous associations with other proteins such as FOXP2. The interactions that YY1 makes with itself, FOXP2 and DNA form part of an intricate mechanism of transcriptional regulation by YY1, which is vital for appropriate neural development.

Insight into Calcium-Binding Motifs of Intrinsically Disordered Proteins.

Motifs within proteins help us categorize their functions. Intrinsically disordered proteins (IDPs) are rich in short linear motifs, conferring them many different roles. IDPs are also frequently highly charged and, therefore, likely to interact with ions. Canonical calcium-binding motifs, such as the EF-hand, often rely on the formation of stabilizing flanking helices, which are a key characteristic of folded proteins, but are absent in IDPs. In this study, we probe the existence of a calcium-binding motif relevant to IDPs. Upon screening several carefully selected IDPs using NMR spectroscopy supplemented with affinity quantification by colorimetric assays, we found calcium-binding motifs in IDPs which could be categorized into at least two groups-an Excalibur-like motif, sequentially similar to the EF-hand loop, and a condensed-charge motif carrying repetitive negative charges. The motifs show an affinity for calcium typically in the ~100 µM range relevant to regulatory functions and, while calcium binding to the condensed-charge motif had little effect on the overall compaction of the IDP chain, calcium binding to Excalibur-like motifs resulted in changes in compaction. Thus, calcium binding to IDPs may serve various structural and functional roles that have previously been underreported.

The biology of tardigrade disordered proteins in extreme stress tolerance.

Disordered proteins have long been known to help mediate tolerance to different abiotic stresses including freezing, osmotic stress, high temperatures, and desiccation in a diverse set of organisms. Recently, three novel families of intrinsically disordered proteins were identified in tardigrades, microscopic animals capable of surviving a battery of environmental extremes. These three families include the Cytoplasmic-, Secreted-, and Mitochondrial- Abundant Heat Soluble (CAHS, SAHS, and MAHS) proteins, which are collectively termed Tardigrade Disordered Proteins (TDPs). At the level of sequence conservation TDPs are unique to tardigrades, and beyond their high degree of disorder the CAHS, SAHS, and MAHS families do not resemble one another. All three families are either highly expressed constitutively, or significantly enriched in response to desiccation. In vivo, ex vivo, and in vitro experiments indicate functional roles for members of each TDP family in mitigating cellular perturbations induced by various abiotic stresses. What is currently lacking is a comprehensive and holistic understanding of the fundamental mechanisms by which TDPs function, and the properties of TDPs that allow them to function via those mechanisms. A quantitative and systematic approach is needed to identify precisely what cellular damage TDPs work to prevent, what sequence features are important for these functions, and how those sequence features contribute to the underlying mechanisms of protection. Such an approach will inform us not only about these fascinating proteins, but will also provide insights into how the sequence of a disordered protein can dictate its functional, structural, and dynamic properties. Video Abstract.

Structural and Biophysical Insights into the Function of the Intrinsically Disordered Myc Oncoprotein.

Myc is a transcription factor driving growth and proliferation of cells and involved in the majority of human tumors. Despite a huge body of literature on this critical oncogene, our understanding of the exact molecular determinants and mechanisms that underlie its function is still surprisingly limited. Indubitably though, its crucial and non-redundant role in cancer biology makes it an attractive target. However, achieving successful clinical Myc inhibition has proven challenging so far, as this nuclear protein is an intrinsically disordered polypeptide devoid of any classical ligand binding pockets. Indeed, Myc only adopts a (partially) folded structure in some contexts and upon interacting with some protein partners, for instance when dimerizing with MAX to bind DNA. Here, we review the cumulative knowledge on Myc structure and biophysics and discuss the implications for its biological function and the development of improved Myc inhibitors. We focus this biophysical walkthrough mainly on the basic region helix-loop-helix leucine zipper motif (bHLHLZ), as it has been the principal target for inhibitory approaches so far.

Characterization of the Binding of Small Molecules to Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) comprise a large fraction of eukaryotic proteomes. IDPs are prevalent in cellular regulation, signaling networks, and disease pathways. The abundance and activity of IDPs is tightly controlled at multiple levels, and their dysregulation is associated with disease. Because of the importance of IDPs in both normal and disease states of the cell, IDPs are attractive targets for modulation by small molecules both to understand their biology and to provide potential drug leads. Multiple screens have successfully identified small molecules that bind to IDPs. Here, we describe how surface plasmon resonance, NMR, and fluorescence methods can be used to characterize the direct binding affinity between small molecules and IDPs. We describe how these techniques can contribute to identifying previously unknown small-molecule binding sites on IDPs.

Effect of hemin, baicalein and heme oxygenase-1 (HO-1) enzyme activity inhibitors on Cd-induced accumulation of HO-1, HSPs and aggresome-like structures in Xenopus kidney epithelial cells.

Cadmium is a highly toxic environmental pollutant that can cause many adverse effects including cancer, neurological disease and kidney damage. Aquatic amphibians are particularly susceptible to this toxicant as it was shown to cause developmental abnormalities and genotoxic effects. In mammalian cells, the accumulation of heme oxygenase-1 (HO-1), which catalyzes the breakdown of heme into CO, free iron and biliverdin, was reported to protect cells against potentially lethal concentrations of CdCl2. In the present study, CdCl2 treatment of A6 kidney epithelial cells, derived from the frog, Xenopus laevis, induced the accumulation of HO-1, heat shock protein 70 (HSP70) and HSP30 as well as an increase in the production of aggregated protein and aggresome-like structures. Treatment of cells with inhibitors of HO-1 enzyme activity, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP), enhanced CdCl2-induced actin cytoskeletal disorganization and the accumulation of HO-1, HSP70, aggregated protein and aggresome-like structures. Treatment of cells with hemin and baicalein, which were previously shown to provide cytoprotection against various stresses, induced HO-1 accumulation in a concentration-dependent manner. Also, treatment of cells with hemin and baicalein suppressed CdCl2-induced actin dysregulation and the accumulation of aggregated protein and aggresome-like structures. This cytoprotective effect was inhibited by SnPP. These results suggest that HO-1-mediated protection against CdCl2 toxicity includes the maintenance of actin cytoskeletal and microtubular structure and the suppression of aggregated protein and aggresome-like structures.

Sesamin extends lifespan through pathways related to dietary restriction in Caenorhabditis elegans.

PURPOSE: Sesamin, a polyphenolic compound found in sesame seeds, has been reported to exert a variety of beneficial health effects. We have previously reported that sesamin increases the lifespan of Caenorhabditis elegans. In this study, we investigated the molecular mechanisms underlying the longevity effect of sesamin in C. elegans. METHODS: Starting from three days of age, Caenorhabditis elegans animals were fed a standard diet alone or supplemented with sesamin. A C. elegans genome array was used to perform a comprehensive expression analysis. Genes that showed differential expression were validated using real-time PCR. Mutant or RNAi-treated animals were fed sesamin, and the lifespan was determined to identify the genes involved in the longevity effects of sesamin. RESULTS: The microarray analysis revealed that endoplasmic reticulum unfolded protein response-related genes, which have been reported to show decreased expression under conditions of SIR-2.1/Sirtuin 1 (SIRT1) overexpression, were downregulated in animals supplemented with sesamin. Sesamin failed to extend the lifespan of sir-2.1 knockdown animals and of sir-2.1 loss-of-function mutants. Sesamin was also ineffective in bec-1 RNAi-treated animals; bec-1 is a key regulator of autophagy, and is necessary for longevity induced by sir-2.1 overexpression. Furthermore, the heterozygotic mutation of daf-15, which encodes the target of rapamycin (TOR)-binding partner Raptor, abolished lifespan extension by sesamin. Moreover, sesamin did not prolong the lifespan of loss-of-function mutants of aak-2, which encodes the AMP-activated protein kinase (AMPK). CONCLUSIONS: Sesamin extends the lifespan of C. elegans through several dietary restriction-related signaling pathways, including processes requiring SIRT1, TOR, and AMPK.

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC's BTPC subunit's at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 µM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKß are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS.

Characterizing the Structure and Oligomerization of Major Royal Jelly Protein 1 (MRJP1) by Mass Spectrometry and Complementary Biophysical Tools.

Royal jelly (RJ) triggers the development of female honeybee larvae into queens. This effect has been attributed to the presence of major royal jelly protein 1 (MRJP1) in RJ. MRJP1 isolated from royal jelly is tightly associated with apisimin, a 54-residue α-helical peptide that promotes the noncovalent assembly of MRJP1 into multimers. No high-resolution structural data are available for these complexes, and their binding stoichiometry remains uncertain. We examined MRJP1/apisimin using a range of biophysical techniques. We also investigated the behavior of deglycosylated samples, as well as samples with reduced apisimin content. Our mass spectrometry (MS) data demonstrate that the native complexes predominantly exist in a (MRJP14 apisimin4) stoichiometry. Hydrogen/deuterium exchange MS reveals that MRJP1 within these complexes is extensively disordered in the range of residues 20-265. Marginally stable secondary structure (likely antiparallel ß-sheet) exists around residues 266-432. These weakly structured regions interchange with conformers that are extensively unfolded, giving rise to bimodal (EX1) isotope distributions. We propose that the native complexes have a "dimer of dimers" quaternary structure in which MRJP1 chains are bridged by apisimin. Specifically, our data suggest that apisimin acts as a linker that forms hydrophobic contacts involving the MRJP1 segment 316VLFFGLV322. Deglycosylation produces large soluble aggregates, highlighting the role of glycans as aggregation inhibitors. Samples with reduced apisimin content form dimeric complexes with a (MRJP12 apisimin1) stoichiometry. The information uncovered in this work will help pave the way toward a better understanding of the unique physiological role played by MRJP1 during queen differentiation.

Treatment of cardiovascular disease by traditional Chinese medicine against pregnane X receptor.

Recently, cardiovascular disease, also known as loop circulatory system diseases or disorders, is one of the serious diseases including heart disease, stroke, atherosclerosis, myocardial infarction, hypertension, hypotension, and thrombosis. Human pregnane X receptor, PXR, plays a crucial role in exogenous and endobiotic metabolism for rabbit, rat, mouse, and human. The PXR activation can protect the blood vessels from damage of hazardous substances. In this study we aim to investigate the potent lead compounds as PXR receptor agonist against cardiovascular disease. To improve drug development of TCM compounds, we aim to investigate the potent lead compounds as PXR agonists from the TCM compounds in TCM Database@Taiwan. The top three TCM compounds, bis(4-hydroxybenzyl) ether mono-ß-D-glucopyranoside (BEMG), ixerisoside, and tangshenoside II, have displayed higher potent binding affinities than the positive control, PNU-142721, in the docking simulation. After MD simulations, which can optimize the result of docking simulation and validate the stability of H-bonds between each ligand and PXR protein under dynamic conditions, top TCM compounds, BEMG and tangshenoside II, maintain most of interactions with PXR protein, which keep the ligand binding stable in the binding domain. Hence, we propose BEMG and tangshenoside II as potential lead compounds for further study in drug development process with the PXR protein.

The inhibition of folylpolyglutamate synthetase (folC) in the prevention of drug resistance in Mycobacterium tuberculosis by traditional Chinese medicine.

Tuberculosis (TB) is an infectious disease caused by many strains of mycobacteria, but commonly Mycobacterium tuberculosis. As a possible method of reducing the drug resistance of M. tuberculosis, this research investigates the inhibition of Folylpolyglutamate synthetase, a protein transcript from the resistance association gene folC. After molecular docking to screen the traditional Chinese medicine (TCM) database, the candidate TCM compounds, with Folylpolyglutamate synthetase, were selected by molecular dynamics. The 10,000 ps simulation in association with RMSD analysis and total energy and structural variation defined the protein-ligand interaction. The selected TCM compounds Saussureamine C, methyl 3-O-feruloylquinate, and Labiatic acid have been found to inhibit the activity of bacteria and viruses and to regulate immunity. We also suggest the possible pathway in protein for each ligand. Compared with the control, similar interactions and structural variations indicate that these compounds might have an effect on Folylpolyglutamate synthetase. Finally, we suggest Saussureamine C is the best candidate compound as the complex has a high score, maintains its structural composition, and has a larger variation value than the control, thus inhibiting the drug resistance ability of Mycobacterium tuberculosis.

A creature with a hundred waggly tails: intrinsically disordered proteins in the ribosome.

Intrinsic disorder (i.e., lack of a unique 3-D structure) is a common phenomenon, and many biologically active proteins are disordered as a whole, or contain long disordered regions. These intrinsically disordered proteins/regions constitute a significant part of all proteomes, and their functional repertoire is complementary to functions of ordered proteins. In fact, intrinsic disorder represents an important driving force for many specific functions. An illustrative example of such disorder-centric functional class is RNA-binding proteins. In this study, we present the results of comprehensive bioinformatics analyses of the abundance and roles of intrinsic disorder in 3,411 ribosomal proteins from 32 species. We show that many ribosomal proteins are intrinsically disordered or hybrid proteins that contain ordered and disordered domains. Predicted globular domains of many ribosomal proteins contain noticeable regions of intrinsic disorder. We also show that disorder in ribosomal proteins has different characteristics compared to other proteins that interact with RNA and DNA including overall abundance, evolutionary conservation, and involvement in protein-protein interactions. Furthermore, intrinsic disorder is not only abundant in the ribosomal proteins, but we demonstrate that it is absolutely necessary for their various functions.

On the importance of polar interactions for complexes containing intrinsically disordered proteins.

There is a growing recognition for the importance of proteins with large intrinsically disordered (ID) segments in cell signaling and regulation. ID segments in these proteins often harbor regions that mediate molecular recognition. Coupled folding and binding of the recognition regions has been proposed to confer high specificity to interactions involving ID segments. However, researchers recently questioned the origin of the interaction specificity of ID proteins because of the overrepresentation of hydrophobic residues in their interaction interfaces. Here, we focused on the role of polar and charged residues in interactions mediated by ID segments. Making use of the extended nature of most ID segments when in complex with globular proteins, we first identified large numbers of complexes between globular proteins and ID segments by using radius-of-gyration-based selection criteria. Consistent with previous studies, we found the interfaces of these complexes to be enriched in hydrophobic residues, and that these residues contribute significantly to the stability of the interaction interface. However, our analyses also show that polar interactions play a larger role in these complexes than in structured protein complexes. Computational alanine scanning and salt-bridge analysis indicate that interfaces in ID complexes are highly complementary with respect to electrostatics, more so than interfaces of globular proteins. Follow-up calculations of the electrostatic contributions to the free energy of binding uncovered significantly stronger Coulombic interactions in complexes harbouring ID segments than in structured protein complexes. However, they are counter-balanced by even higher polar-desolvation penalties. We propose that polar interactions are a key contributing factor to the observed high specificity of ID segment-mediated interactions.