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The development of aging is associated with the disruption of key cellular processes manifested as well-established hallmarks of aging. Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) have no stable tertiary structure that provide them a power to be configurable hubs in signaling cascades and regulate many processes, potentially including those related to aging. There is a need to clarify the roles of IDPs/IDRs in aging. The dataset of 1702 aging-related proteins was collected from established aging databases and experimental studies. There is a noticeable presence of IDPs/IDRs, accounting for about 36% of the aging-related dataset, which is however less than the disorder content of the whole human proteome (about 40%). A Gene Ontology analysis of the used here aging proteome reveals an abundance of IDPs/IDRs in one-third of aging-associated processes, especially in genome regulation. Signaling pathways associated with aging also contain IDPs/IDRs on different hierarchical levels, revealing the importance of "structure-function continuum" in aging. Protein–protein interaction network analysis showed that IDPs present in different clusters associated with different aging hallmarks. Protein cluster with IDPs enrichment has simultaneously high liquid–liquid phase separation (LLPS) probability, “nuclear” localization and DNA-associated functions, related to aging hallmarks: genomic instability, telomere attrition, epigenetic alterations, and stem cells exhaustion. Intrinsic disorder, LLPS, and aggregation propensity should be considered as features that could be markers of pathogenic proteins. Overall, our analyses indicate that IDPs/IDRs play significant roles in aging-associated processes, particularly in the regulation of DNA functioning. IDP aggregation, which can lead to loss of function and toxicity, could be critically harmful to the cell. A structure-based analysis of aging and the identification of proteins that are particularly susceptible to disturbances can enhance our understanding of the molecular mechanisms of aging and open up new avenues for slowing it down. [ABSTRACT FROM AUTHOR]

The human MDM2 protein regulates the tumor suppressor protein p53 by restricting its transcriptional activity and by promoting p53 degradation. MDM2 is ubiquitously expressed, with its overexpression implicated in many forms of cancer. The inhibitory effects of MDM2 on p53 have been shown to involve its N-terminal p53-binding domain and its C-terminal RING domain. The presence of an intact central acidic domain of MDM2 has also been shown to regulate p53 ubiquitination, with this domain shown to directly interact with the p53 DNA-binding domain to regulate the DNA binding activity of p53. To date, little structural information has been obtained for the MDM2 acidic domain. Thus, to gain insight into the structure and function relationship of this region, we have applied solution-state NMR spectroscopy to characterize the segment of MDM2 spanning residues 215–300. These boundaries for the acidic domain were determined on the basis of consensus observed in multiple sequence alignment. Here, we report the 1H, 15N and 13C backbone and 13Cβ chemical shift assignments and steady-state {1H}-15N heteronuclear NOE enhancement factors as a function of residue for the acidic domain of MDM2. We show that this domain exhibits the hallmarks of being a disordered protein, on the basis both of assigned chemical shifts and residue-level backbone dynamics, with localized variation in secondary structure propensity inferred from chemical shift analysis. [ABSTRACT FROM AUTHOR]

Intrinsically disordered regions in proteins have been shown to be important in protein function. However, not all proteins contain the same amount of intrinsic disorder. The variation in the levels of intrinsic disorder in different types of proteins has been extensively studied over the last two decades. It is now known that the levels of intrinsic disorder vary in proteins across organisms, functions, diseases, and cellular locations. This review consolidates the known trends in the abundance of intrinsic disorder identified in groups of proteins across varying conditions and functions. It also presents new data towards the understanding of intrinsic disorder in cell type-specific proteins. [ABSTRACT FROM AUTHOR]

IncC from the low-copy number plasmid RK2, is a member of the ParA family of proteins required for partitioning DNA in many bacteria and plasmids. It is an ATPase that binds DNA and its ParB protein partner, KorB. Together, the proteins move replicated DNA to appropriate cellular positions, so that each daughter cell inherits a copy on cell division. IncC from RK2 is expressed in two forms. IncC2 is homologous to bacterial ParA proteins, while IncC1 has an N-terminal extension of 105 amino acids and is similar in length to ParA homologues in other plasmids. We have been examining the role of this extension, here called IncC NTD. We present its backbone NMR chemical shift assignments and show that it is entirely intrinsically disordered. The assignments were achieved using C-detected, CON-based spectra, complemented by HNN spectra to obtain connectivities from three adjacent amino acids. We also observed evidence of deamidation of the protein at a GNGG sequence, to give isoAsp, giving 2 sets of peaks for residues up to 5 amino acids on either side of the modification. We have assigned resonances from around the position of modification for this form of the protein. [ABSTRACT FROM AUTHOR]

Human Hippo signaling pathway plays an important role in the tumorigenesis of diverse cancers and has been recognized as an attractive therapeutic target of gastric cancer. The transcriptional coactivators Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) are the major downstream effectors of Hippo, which interact primarily with transcriptional enhanced associate domain (TEAD) protein through their TEAD-binding domain (TBD). Competitive disruption of the TEAD–YAP/TAZ interaction using peptide inhibitors has been exploited as a potential strategy to treat gastric cancer by regulating Hippo signaling. Here, the crystal structures of TEAD complex with YAP/TAZ TBD domain are investigated systematically at structural, energetic and dynamic levels, from which two binding hotspots are identified; they separately correspond to an α-helix and a Ω-loop of TBD domain, and contribute essentially to the complex interaction. Several linear peptide segments derived from the hotspot regions are highly flexibility and exhibit moderate or modest affinity for TEAD. The Ω-loop-derived peptides are found to have a higher affinity, which are cyclized by introducing a disulfide bridge across their two termini. Affinity assay confirms that the cyclization can considerably improve peptide affinity by 3.7–6.6-fold. Computational analysis reveals that the designed cyclic peptides exhibit a decreased flexibility and intrinsic disorder; they can roughly maintain in native active conformation out of TBD protein context, with a reduced entropy cost upon binding to TEAD. [ABSTRACT FROM AUTHOR]

Intrinsic disorder can be found in all proteomes of all kingdoms of life and in viruses, being particularly prevalent in the eukaryotes. We conduct a comprehensive analysis of the intrinsic disorder in the human proteins while mapping them into 24 compartments of the human cell. In agreement with previous studies, we show that human proteins are significantly enriched in disorder relative to a generic protein set that represents the protein universe. In fact, the fraction of proteins with long disordered regions and the average protein-level disorder content in the human proteome are about 3 times higher than in the protein universe. Furthermore, levels of intrinsic disorder in the majority of human subcellular compartments significantly exceed the average disorder content in the protein universe. Relative to the overall amount of disorder in the human proteome, proteins localized in the nucleus and cytoskeleton have significantly increased amounts of disorder, measured by both high disorder content and presence of multiple long intrinsically disordered regions. We empirically demonstrate that, on average, human proteins are assigned to 2.3 subcellular compartments, with proteins localized to few subcellular compartments being more disordered than the proteins that are localized to many compartments. Functionally, the disordered proteins localized in the most disorder-enriched subcellular compartments are primarily responsible for interactions with nucleic acids and protein partners. This is the first-time disorder is comprehensively mapped into the human cell. Our observations add a missing piece to the puzzle of functional disorder and its organization inside the cell. [ABSTRACT FROM AUTHOR]

Orphan genes, lacking detectable homologs in outgroup species, typically represent 10–30% of eukaryotic genomes. Efforts to find the source of these young genes indicate that de novo emergence from non-coding DNA may in part explain their prevalence. Here, we investigate the roots of orphan gene emergence in the Drosophila genus. Across the annotated proteomes of twelve species, we find 6297 orphan genes within 4953 taxon-specific clusters of orthologs. By inferring the ancestral DNA as non-coding for between 550 and 2467 (8.7–39.2%) of these genes, we describe for the first time how de novo emergence contributes to the abundance of clade-specific Drosophila genes. In support of them having functional roles, we show that de novo genes have robust expression and translational support. However, the distinct nucleotide sequences of de novo genes, which have characteristics intermediate between intergenic regions and conserved genes, reflect their recent birth from non-coding DNA. We find that de novo genes encode more disordered proteins than both older genes and intergenic regions. Together, our results suggest that gene emergence from non-coding DNA provides an abundant source of material for the evolution of new proteins. Following gene birth, gradual evolution over large evolutionary timescales moulds sequence properties towards those of conserved genes, resulting in a continuum of properties whose starting points depend on the nucleotide sequences of an initial pool of novel genes. [ABSTRACT FROM AUTHOR]

Computational prediction of intrinsic disorder in protein sequences dates back to late 1970 and has flourished in the last two decades. We provide a brief historical overview, and we review over 30 recent predictors of disorder. We are the first to also cover predictors of molecular functions of disorder, including 13 methods that focus on disordered linkers and disordered protein-protein, protein-RNA, and protein-DNA binding regions. We overview their predictive models, usability, and predictive performance. We highlight newest methods and predictors that offer strong predictive performance measured based on recent comparative assessments. We conclude that the modern predictors are relatively accurate, enjoy widespread use, and many of them are fast. Their predictions are conveniently accessible to the end users, via web servers and databases that store pre-computed predictions for millions of proteins. However, research into methods that predict many not yet addressed functions of intrinsic disorder remains an outstanding challenge. [ABSTRACT FROM AUTHOR]

Conformational flexibility conferred though regions of intrinsic structural disorder allows proteins to behave as dynamic molecules. While it is well-known that intrinsically disordered regions can undergo disorder-to-order transitions in real-time as part of their function, we also are beginning to learn more about the dynamics of disorder-to-order transitions along evolutionary time-scales. Intrinsically disordered regions endow proteins with functional promiscuity, which is further enhanced by the ability of some of these regions to undergo real-time disorder-to-order transitions. Disorder content affects gene retention after whole genome duplication, but it is not necessarily conserved. Altered patterns of disorder resulting from evolutionary disorder-to-order transitions indicate that disorder evolves to modify function through refining stability, regulation, and interactions. Here, we review the evolution of intrinsically disordered regions in eukaryotic proteins. We discuss the interplay between secondary structure and disorder on evolutionary time-scales, the importance of disorder for eukaryotic proteome expansion and functional divergence, and the evolutionary dynamics of disorder. [ABSTRACT FROM AUTHOR]

There is ample evidence that many proteins or regions of proteins lack a well-defined folded structure under native-like conditions. These are called intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs). Whether this intrinsic disorder is also their main structural characteristic in living cells has been a matter of intense debate. The structural analysis of IDPs became an important challenge also because of their involvement in a plethora of human diseases, which made IDPs attractive targets for therapeutic development. Therefore, biophysical approaches are increasingly being employed to probe the structural and dynamical state of proteins, not only in isolation in a test tube, but also in a complex biological environment and even within intact cells. Here, we survey direct and indirect evidence that structural disorder is in fact the physiological state of many proteins in the proteome. The paradigmatic case of α-synuclein is used to illustrate the controversial nature of this topic. [ABSTRACT FROM AUTHOR]

The focus of the computational structural biology community has taken a dramatic shift over the past one-and-a-half decades from the classical protein structure prediction problem to the possible understanding of intrinsically disordered proteins (IDP) or proteins containing regions of disorder (IDPR). The current interest lies in the unraveling of a disorder-to-order transitioning code embedded in the amino acid sequences of IDPs/IDPRs. Disordered proteins are characterized by an enormous amount of structural plasticity which makes them promiscuous in binding to different partners, multi-functional in cellular activity and atypical in folding energy landscapes resembling partially folded molten globules. Also, their involvement in several deadly human diseases (e.g. cancer, cardiovascular and neurodegenerative diseases) makes them attractive drug targets, and important for a biochemical understanding of the disease(s). The study of the structural ensemble of IDPs is rather difficult, in particular for transient interactions. When bound to a structured partner, an IDPR adapts an ordered conformation in the complex. The residues that undergo this disorder-to-order transition are called protean residues, generally found in short contiguous stretches and the first step in understanding the modus operandi of an IDP/IDPR would be to predict these residues. There are a few available methods which predict these protean segments from their amino acid sequences; however, their performance reported in the literature leaves clear room for improvement. With this background, the current study presents 'Proteus', a random forest classifier that predicts the likelihood of a residue undergoing a disorder-to-order transition upon binding to a potential partner protein. The prediction is based on features that can be calculated using the amino acid sequence alone. Proteus compares favorably with existing methods predicting twice as many true positives as the second best method (55 vs. 27%) with a much higher precision on an independent data set. The current study also sheds some light on a possible 'disorder-to-order' transitioning consensus, untangled, yet embedded in the amino acid sequence of IDPs. Some guidelines have also been suggested for proceeding with a real-life structural modeling involving an IDPR using Proteus. [ABSTRACT FROM AUTHOR]

Collapsin response mediator protein 2 (CRMP-2) is a neuronal protein involved in axonal pathfinding. Intense research is focusing on its role in various neurological diseases. Despite a wealth of studies, not much is known about the molecular mechanisms of CRMP-2 function in vivo. The detailed structure-function relationships of CRMP-2 have also largely remained unknown, in part due to the fact that the available crystal structures lack the C-terminal tail, which is known to be a target for many post-translational modifications and protein interactions. Although CRMP-2, and other CRMPs, belong to the dihydropyrimidinase family, they have lost the enzymatic active site. Drug candidates for CRMP-2-related processes have come up during the recent years, but no reports of CRMP-2 complexes with small molecules have emerged. Here, CRMP-2 was studied at 1.25-Å resolution using X-ray crystallography. In addition, ligands were docked into the homotetrameric structure, and the C-terminal tail of CRMP-2 was produced recombinantly and analyzed. We have obtained the human CRMP-2 crystal structure at atomic resolution and could identify small-molecule binding pockets in the protein. Structures obtained in different crystal forms highlight flexible regions near possible ligand-binding pockets. We also used the CRMP-2 structure to analyze known or suggested post-translational modifications at the 3D structural level. The high-resolution CRMP-2 structure was also used for docking experiments with the sulfur amino acid metabolite lanthionine ketimine and its ester. We show that the C-terminal tail is intrinsically disordered, but it has conserved segments that may act as interaction sites. Our data provide the most accurate structural data on CRMPs to date and will be useful in further computational and experimental studies on CRMP-2, its function, and its binding to small-molecule ligands. [ABSTRACT FROM AUTHOR]

H1 linker histones are involved both in the maintenance of chromatin higher-order structure and in gene regulation. H1 binds to linker DNA regions on the surface of the nucleosome. In higher eukaryotes, H1 contains three distinct domains: a short N-terminal domain (NTD), a central globular domain, and a long C-terminal domain (CTD). Terminal domains determine subtype specificity and to a large extent the linker DNA binding and chromatin condensing properties of histone H1. This review is focused on the recent numerous studies that have provided insights in the role of H1 terminal domains in chromatin dynamics. The N- and C-terminal domains behave as intrinsically disordered proteins with coupled binding and folding. We examine the potential kinetic advantages of intrinsic disorder in the recognition of the specific H1 binding sites in chromatin. As typical intrinsically disordered regions, H1 terminal domains are post-translationally modified. Post-translational modifications in the NTD determine the interaction of histone H1 with other proteins involved in heterochromatin formation and transcriptional regulation, while phosphorylation by cyclin-dependent kinases modulates the secondary structure of the CTD and chromatin condensation. We review the arguments in favor of the involvement of H1 hyperphosphorylation in metaphase chromatin condensation and of partial phosphorylation in interphase chromatin relaxation. In addition, the interplay of histone H1 and other chromatin architectural proteins, such as proteins of the high-mobility group, protamines, and MeCP2, is associated with changes in chromatin structure. [ABSTRACT FROM AUTHOR]

Heat-shock protein (HSP)-based immunotherapy is believed to be a promising area of development for cancer treatment as such therapy is characterized by a unique approach to every tumour. It was shown that by inhibition of HSPs it is possible to induce apoptotic cell death in cancer cells. Interestingly, there are a great number of disordered regions in proteins associated with cancer, cardiovascular and neurodegenerative diseases, signalling, and diabetes. HSPs and some specific enzymes were shown to have these disordered regions in their primary structures. The experimental studies of HSPs confirmed that their intrinsically disordered (ID) regions are of functional importance. These ID regions play crucial roles in regulating the specificity of interactions between dimer complexes and their interacting partners. Because HSPs are overexpressed in cancer, predicting the locations of ID regions and binding sites in these proteins will be important for developing novel cancer therapeutics. In our previous studies, signal processing methods have been successfully used for protein structure-function analysis (i.e. for determining functionally important amino acids and the locations of protein active sites). In this paper, we present and discuss a novel approach for predicting the locations of ID regions in the selected cancer-related HSPs. [ABSTRACT FROM AUTHOR]

Abstract Recent studies have emphasized the value of including structural information into the topological analysis of protein networks. Here, we utilized structural information to investigate the role of intrinsic disorder in these networks. Hub proteins tend to be more disordered than other proteins (i.e. the proteome average); however, we find this only true for those with one or two binding interfaces (‘single’‐interface hubs). In contrast, the distribution of disordered residues in multi‐interface hubs is indistinguishable from the overall proteome. Surprisingly, we find that the binding interfaces in single‐interface hubs are highly structured, as is the case for multi‐interface hubs. However, the binding partners of single‐interface hubs tend to have a higher level of disorder than the proteome average, suggesting that their binding promiscuity is related to the disorder of their binding partners. In turn, the higher level of disorder of single‐interface hubs can be partly explained by their tendency to bind to each other in a cascade. A good illustration of this trend can be found in signaling pathways and, more specifically, in kinase cascades. Finally, our findings have implications for the current controversy related to party and date‐hubs.

In higher eukaryotes, proteomes are typically larger than corresponding genomes mostly due to alternative spicing (AS) which is affecting more than 86 % of human genes. Tissue specificity of many proteins is often determined by the AS of pre-mRNAs that generates multiple proteins from a single gene. Aberrations in AS are found in numerous human diseases. This article shows that AS expanded the classic 'one-gene-one-protein' paradigm to the 'one-gene-many-proteins' concept. AS is intimately associated with protein intrinsic disorder. There is a tight connection between the altered AS of some key intrinsically disordered proteins and pathogenesis of neurodegenerative diseases, cardiovascular disease, cancer, and diabetes. The development of drugs for splice variants is a challenging endeavor. AS has tremendous influence on the applicability of several drugs developed to affect functions typically attributed to the ordered parts of disease-related proteins. Although there are several means for the differential targeting of splice variants, new approaches are obviously needed. It is expected that better understanding of the molecular mechanisms underlying modulation of biological activities of numerous spliced variants will be achieved and new drug design approaches for proteins with multiple splice variants will be developed. [ABSTRACT FROM AUTHOR]

PsbP (23 kDa) is an extrinsic eukaryotic protein of photosystem II found in the thylakoid membrane of higher plants and green algae. It has been proven to be indispensable for proper functioning of the oxygen evolving complex. By interaction with other extrinsic proteins (PsbQ, PsbO and PsbR), it modulates the concentration of two cofactors of the water splitting reaction, Ca and Cl. The crystallographic structure of PsbP from Spinacia oleracea lacks the N-terminal part as well as two inner regions which were modelled as loops. Those unresolved parts are believed to be functionally crucial for the binding of PsbP to the thylakoid membrane. In this NMR study we report H, N and C resonance assignments of the backbone and side chain atoms of the PsbP protein. Based on these data, an estimate of the secondary structure has been made. The structural motifs found fit the resolved parts of the crystallographic structure very well. In addition, the complete assignment set provides preliminary insight into the dynamic regions. [ABSTRACT FROM AUTHOR]

Allostery is a biological regulation mechanism of significant importance in cell signaling, metabolism, and disease. Although the ensemble basis of allostery has been known for years, only recently has emphasis shifted from interpreting allosteric mechanism in terms of discrete structural pathways to ones that focus on the statistical nature of the signal propagation process, providing a vehicle to unify allostery in structured, dynamic, and disordered systems. In particular, intrinsically disordered (ID) proteins (IDPs), which lack a unique, stable structure, have been directly demonstrated to exhibit allostery in numerous systems, a reality that challenges traditional structure-based models that focus on allosteric pathways. In this chapter, we will discuss the historical context of allostery and focus on studies from human glucocorticoid receptor (GR), a member of the steroid hormone receptor (SHR) family. The numerous translational isoforms of the disordered N-terminal domain of GR consist of coupled thermodynamic domains that contribute to the delicate balance of states in the ensemble and hence in vivo activity. The data are quantitatively interpreted using the ensemble allosteric model (EAM) that considers only the intrinsic and measurable energetics of allosteric systems. It is demonstrated that the EAM provides mechanistic insight into the distribution of states in solution and provides an interpretation for how certain translational isoforms of GR display enhanced and repressed transcriptional activities. The ensemble nature of allostery illuminated from these studies lends credence to the EAM and provides ground rules for allostery in all systems. [ABSTRACT FROM AUTHOR]

Recent years witnessed increased interest in intrinsically disordered proteins and regions. These proteins and regions are abundant and possess unique structural features and a broad functional repertoire that complements ordered proteins. However, modern studies on the abundance and functions of intrinsically disordered proteins and regions are relatively limited in size and scope of their analysis. To fill this gap, we performed a broad and detailed computational analysis of over 6 million proteins from 59 archaea, 471 bacterial, 110 eukaryotic and 325 viral proteomes. We used arguably more accurate consensus-based disorder predictions, and for the first time comprehensively characterized intrinsic disorder at proteomic and protein levels from all significant perspectives, including abundance, cellular localization, functional roles, evolution, and impact on structural coverage. We show that intrinsic disorder is more abundant and has a unique profile in eukaryotes. We map disorder into archaea, bacterial and eukaryotic cells, and demonstrate that it is preferentially located in some cellular compartments. Functional analysis that considers over 1,200 annotations shows that certain functions are exclusively implemented by intrinsically disordered proteins and regions, and that some of them are specific to certain domains of life. We reveal that disordered regions are often targets for various post-translational modifications, but primarily in the eukaryotes and viruses. Using a phylogenetic tree for 14 eukaryotic and 112 bacterial species, we analyzed relations between disorder, sequence conservation and evolutionary speed. We provide a complete analysis that clearly shows that intrinsic disorder is exceptionally and uniquely abundant in each domain of life. [ABSTRACT FROM AUTHOR]

Post-translational modification of protein lysines was recently shown to be a common feature of eukaryotic organisms. The ubiquitin modification is regarded as a versatile regulatory mechanism with many important cellular roles. Large-scale datasets are becoming available for H. sapiens ubiquitination. However, using current experimental techniques the vast majority of their sites remain unidentified and in silico tools may offer an alternative. Here, we introduce Rapid UBIquitination (RUBI) a sequence-based ubiquitination predictor designed for rapid application on a genome scale. RUBI was constructed using an iterative approach. At each iteration, important factors which influenced performance and its usability were investigated. The final RUBI model has an AUC of 0.868 on a large cross-validation set and is shown to outperform other available methods on independent sets. Predicted intrinsic disorder is shown to be weakly anti-correlated to ubiquitination for the H. sapiens dataset and improves performance slightly. RUBI predicts the number of ubiquitination sites correctly within three sites for ca. 80 % of the tested proteins. The average potentially ubiquitinated proteome fraction is predicted to be at least 25 % across a variety of model organisms, including several thousand possible H. sapiens proteins awaiting experimental characterization. RUBI can accurately predict ubiquitination on unseen examples and has a signal across different eukaryotic organisms. The factors which influenced the construction of RUBI could also be tested in other post-translational modification predictors. One of the more interesting factors is the influence of intrinsic protein disorder on ubiquitinated lysines where residues with low disorder probability are preferred. [ABSTRACT FROM AUTHOR]

Kinesin molecular motors perform a myriad of intracellular transport functions. While their mechanochemical mechanisms are well understood and well-conserved throughout the superfamily, the cargo-binding and regulatory mechanisms governing the activity of kinesins are highly diverse and, in general, incompletely characterized. Here we present evidence from bioinformatic predictions indicating that most kinesin superfamily members contain significant regions of intrinsically disordered (ID) residues. ID regions can bind to multiple partners with high specificity and are highly labile to post-translational modification and degradation signals. In kinesins, the predicted ID regions are primarily found in areas outside the motor domains, where primary sequences diverge by family, suggesting that the ID may be a critical structural element for determining the functional specificity of individual kinesins. To support this concept, we present a systematic analysis of the kinesin superfamily, family by family, for predicted ID regions. We combine this analysis with a comprehensive review of kinesin-binding partners and post-translational modifications. We find two key trends across the entire kinesin superfamily. First, ID residues tend to be in the tail regions of kinesins, opposite the superfamily-conserved motor domains. Second, predicted ID regions correlate to regions that are known to bind to cargoes and/or undergo post-translational modifications. We therefore propose that the ID residue is a structural element utilized by the kinesin superfamily in order to impart functional specificity to individual kinesins. [ABSTRACT FROM AUTHOR]

Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca-saturated calmodulin (Ca/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin-radixin-moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G SRGISRFIPPWLKKQKS peptide (pepG) mediates Ca/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G. [ABSTRACT FROM AUTHOR]

Expression of the gene encoding the maize glycine-rich RNA-binding protein MA16 is developmentally regulated and it is involved in environmental stress responses. The MA16 protein shows a wide spectrum of RNA-binding activities. On the basis of in vivo labelling, where a [P]phosphate label was linked to the MA16 protein, Freire and Pages (Plant Mol Biol 29:797-807, ) suggested that the protein may be post-translationally modified by phosphorylation. However, further analysis showed that the [P]phosphate label was sensitive to different treatments, suggesting that modification distinct from protein phosphorylation might occur in the MA16 protein. Biochemical analysis revealed that this [P]phosphate labelling was resistant to phenol extraction and denaturing SDS-PAGE but sensitive to micrococcal nuclease, RNase A and RNase T1 treatments. The mobility of [S] labelled MA16 protein on SDS-PAGE did not significantly changed after the nuclease treatments suggesting that the [P]phosphate label associated to MA16 protein could be a ribonucleotide or a very short ribonucleotide chain. In addition, immunoprecipitation of labelled extracts showed that the ribonucleotide(s) linked to the MA16 protein was removed by phosphorolytic activity. This activity could be catalysed by a phosphate-dependent ribonuclease. The C-terminus of MA16 protein harbouring a glycine-rich domain was predicted to be an intrinsically disordered region. [ABSTRACT FROM AUTHOR]

The discovery of intrinsically disordered proteins (IDP) (i.e., biologically active proteins that do not possess stable secondary and/or tertiary structures) came as an unexpected surprise, as the existence of such proteins is in contradiction to the traditional “sequence→structure→function” paradigm. Accurate prediction of a protein's predisposition to be intrinsically disordered is a necessary prerequisite for the further understanding of principles and mechanisms of protein folding and function, and is a key for the elaboration of a new structural and functional hierarchy of proteins. Therefore, prediction of IDPs has attracted the attention of many researchers, and a number of prediction tools have been developed. Predictions of disorder, in turn, are playing major roles in directing laboratory experiments that are leading to the discovery of ever more disordered proteins, and thereby leading to a positive feedback loop in the investigation of these proteins. In this review of algorithms for intrinsic disorder prediction, the basic concepts of various prediction methods for IDPs are summarized, the strengths and shortcomings of many of the methods are analyzed, and the difficulties and directions of future development of IDP prediction techniques are discussed.Cell Research (2009) 19:929–949. doi: 10.1038/cr.2009.87; published online 14 July 2009 [ABSTRACT FROM AUTHOR]

The five articles in this multi-author review in CMLS provide examples of multi-functionality of proteins belonging to several families. Distinct structural features of proteins suggesting multi-functionality are emphasized: intrinsically disordered elements that can "mold" themselves to fit various binding partners, as well as short linear motifs within larger proteins that perform particular functions. Although only a few protein families are discussed in detail, the conclusions apply to numerous, if not all, proteins. Multi-functionality of virtually every protein implies that the manipulation of its expression levels by over-expression, knockdown, or knockout affects every one of its functions, known and unknown, so that the results of these experiments must be interpreted with this complexity in mind. Particular functions in a multi-functional protein are often fulfilled by identifiable smaller elements that can be expressed separately. Identification of mono-functional elements of a multi-functional protein paves the way to the construction of novel precisely targeted molecular tools for selective manipulation of cellular signaling that can be used for mechanistic studies in cell biology, as well as for therapeutic purposes. [ABSTRACT FROM AUTHOR]

The xenobiotic fluoride is a detrimental toxin which is exported by a double-barrelled fluoride channel (Fluc) in prokaryotes and a single-barrelled fluoride exporter (FEX) in lower eukaryotes. The presence of FEX gene in higher plant species has not been reported till date. In this communication, we have identified FEXs across nine plant species belonging to five different families. Homology modelling predicted the sequential and structural conservation of crucial residues in plant FEXs. It was predicted that the N-terminal segments were the main target sites for phosphorylation. Other post-translational modifications included Arg methylation, Lys acetylation and Cys S-nitrosylation, all of which contribute to protein stability and function. FEXs were predicted to contain lipid moieties which anchor the transporters to the plasma membrane. The crystallizability along with associated properties of plant FEXs was also analyzed to facilitate future experimental analyses. The predicted RNA structure of OsFEX formed several pseudoknots, though no riboswitches could be detected from our prediction. The analysis of upstream regions of FEX in representative plants represented that this gene could be responsive to phytohormone-mediated signaling. This is the first novel report highlighting that FEX genes in plants are probably expressed in a tissue-specific pattern and possibly via a phytohormone-dependent pathway to encode functional FEX proteins during fluoride stress. [ABSTRACT FROM AUTHOR]