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T cell receptor beta (TRB) sequences were recovered from the Cancer Genome Atlas Uveal Melanoma blood exome files. Intrinsic disorder scores for amino acid (AA) sequences of the entire TRB variable region were obtained and evaluated as potentially representative of overall survival (OS) distinctions, i.e., for cases representing the upper and lower 50th percentiles for intrinsic disorder scores. Analyses using four intrinsic disorder assessment tools indicated that a lower intrinsic disorder of the blood-sourced TRB variable regions, including continuous AA sequences of the V-gene segment, the complementarity-determining region-3, and the J-gene segment, was associated with a better OS probability (with log-rank p-values ranging from 0.002 to 0.014). We further determined that intrinsic disorder assessments could be used for OS stratification for a second, immunologically cold cancer: MYCN amplified neuroblastoma. Thus, intrinsic disorder assessments of blood-sourced, full TRB variable regions may provide a novel patient stratification approach for patients with immunologically cold cancers. [ABSTRACT FROM AUTHOR]

Compositionally biased regions (CBRs), ie, tracts that are dominated by a subset of residue types, are common features of eukaryotic proteins. These are often found bounded within or almost coterminous with intrinsically disordered or 'natively unfolded' parts. Here, it is investigated how the function of such intrinsically disordered compositionally biased regions (ID-CBRs) is directly linked to their compositional traits, focusing on the well-characterized yeast (Saccharomyces cerevisiae) proteome as a test case. The ID-CBRs that are clustered together using compositional distance are discovered to have clear functional linkages at various levels of diversity. The specific case of the Sup35p and Rnq1p proteins that underlie causally linked prion phenomena ([PSI+] and [RNQ+]) is highlighted. Their prion-forming ID-CBRs are typically clustered very close together indicating some compositional engendering for [RNQ+] seeding of [PSI+] prions. Delving further, ID-CBRs with distinct types of residue patterning such as 'blocking' or relative segregation of residues into homopeptides are found to have significant functional trends. Specific examples of such ID-CBR functional linkages that are discussed are: Q/N-rich ID-CBRs linked to transcriptional coactivation, S-rich to transcription-factor binding, R-rich to DNA-binding, S/E-rich to protein localization, and D-rich linked to chromatin remodelling. These data may be useful in informing experimental hypotheses for proteins containing such regions. [ABSTRACT FROM AUTHOR]

Orphan nuclear estrogen‐related receptor γ (ERRγ) has been recognized as a potential therapeutic target for cancer, inflammation and metabolic disorder. The ERRγ contains a regulatory AF2 helical tail linked C‐terminally to its ligand‐binding domain (LBD), which is a self‐binding peptide (SBP) and serves as molecular switch to dynamically regulate the receptor alternation between active and inactive states by binding to and unbinding from the AF2‐binding site on ERRγ LBD surface, respectively. Traditional ERRγ modulators are all small‐molecule chemical ligands that can be classified into agonists and inverse agonists in terms of their action mechanism; the agonists stabilize the AF2 in ABS site with an agonist conformation, while the inverse agonists lock the AF2 out of the site to largely abolish ERRγ transcriptional activity. Here, a class of ERRγ peptidic antagonists was described to compete with native AF2 for the ABS site, thus blocking the active state of AF2 binding to ERRγ LBD domain. Self‐inhibitory peptide was derived from the SBP‐covering AF2 region and we expected it can rebind potently to the ABS site by reducing its intrinsic disorder and entropy cost upon the rebinding. Hydrocarbon stapling was employed to do so, which employed an all‐hydrocarbon bridge across the [i, i + 4]‐anchor residue pair in the N‐terminal, middle or C‐terminal region of the self‐inhibitory peptide. As might be expected, it is revealed that the stapled peptides are good binders of ERRγ LBD domain and can effectively compete with the native AF2 helical tail for ERRγ ABS site, which exhibit a basically similar binding mode with AF2 to the site and form diverse noncovalent interactions with the site, thus conferring stability and specificity to the domain–peptide complexes. [ABSTRACT FROM AUTHOR]

Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate‐binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase. [ABSTRACT FROM AUTHOR]

The relationship between pangolin-CoV and SARS-CoV-2 has been a subject of debate. Further evidence of a special relationship between the two viruses can be found by the fact that all known COVID-19 viruses have an abnormally hard outer shell (low M disorder, i.e., low content of intrinsically disordered residues in the membrane (M) protein) that so far has been found in CoVs associated with burrowing animals, such as rabbits and pangolins, in which transmission involves virus remaining in buried feces for a long time. While a hard outer shell is necessary for viral survival, a harder inner shell could also help. For this reason, the N disorder range of pangolin-CoVs, not bat-CoVs, more closely matches that of SARS-CoV-2, especially when Omicron is included. The low N disorder (i.e., low content of intrinsically disordered residues in the nucleocapsid (N) protein), first observed in pangolin-CoV-2017 and later in Omicron, is associated with attenuation according to the Shell-Disorder Model. Our experimental study revealed that pangolin-CoV-2017 and SARS-CoV-2 Omicron (XBB.1.16 subvariant) show similar attenuations with respect to viral growth and plaque formation. Subtle differences have been observed that are consistent with disorder-centric computational analysis. [ABSTRACT FROM AUTHOR]

The human Vitamin K Epoxide Reductase Complex (hVKORC1), a key enzyme that converts vitamin K into the form necessary for blood clotting, requires for its activation the reducing equivalents supplied by its redox partner through thiol–disulphide exchange reactions. The functionally related molecular complexes assembled during this process have never been described, except for a proposed de novo model of a 'precursor' complex of hVKORC1 associated with protein disulphide isomerase (PDI). Using numerical approaches (in silico modelling and molecular dynamics simulation), we generated alternative 3D models for each molecular complex bonded either covalently or non-covalently. These models differ in the orientation of the PDI relative to hVKORC1 and in the cysteine residue involved in forming protein–protein disulphide bonds. Based on a comparative analysis of these models' shape, folding, and conformational dynamics, the most probable putative complexes, mimicking the 'precursor', 'intermediate', and 'successor' states, were suggested. In addition, we propose using these complexes to develop the 'allo-network drugs' necessary for treating blood diseases. [ABSTRACT FROM AUTHOR]

Disordered linkers (DLs) are intrinsically disordered regions that facilitate movement between adjacent functional regions/domains, contributing to many key cellular functions. The recently completed second Critical Assessments of protein Intrinsic Disorder prediction (CAID2) experiment evaluated DL predictions by considering a rather narrow scenario when predicting 40 proteins that are already known to have DLs. We expand this evaluation by using a much larger set of nearly 350 test proteins from CAID2 and by investigating three distinct scenarios: (1) prediction residues in DLs vs. in non-DL regions (typical use of DL predictors); (2) prediction of residues in DLs vs. other disordered residues (to evaluate whether predictors can differentiate residues in DLs from other types of intrinsically disordered residues); and (3) prediction of proteins harboring DLs. We find that several methods provide relatively accurate predictions of DLs in the first scenario. However, only one method, APOD, accurately identifies DLs among other types of disordered residues (scenario 2) and predicts proteins harboring DLs (scenario 3). We also find that APOD's predictive performance is modest, motivating further research into the development of new and more accurate DL predictors. We note that these efforts will benefit from a growing amount of training data and the availability of sophisticated deep network models and emphasize that future methods should provide accurate results across the three scenarios. [ABSTRACT FROM AUTHOR]

Proteins, especially of eukaryotes, often have disordered domains and may contain multiple folded domains whose relative spatial arrangement is distributed. The MMMx ensemble modeling and analysis toolbox (https://github.com/gjeschke/MMMx) can support the design of experiments to characterize the distributed structure of such proteins, starting from AlphaFold2 predictions or folded domain structures. Weak order can be analyzed with reference to a random coil model or to peptide chains that match the residue‐specific Ramachandran angle distribution of the loop regions and are otherwise unrestrained. The deviation of the mean square end‐to‐end distance of chain sections from their average over sections of the same sequence length reveals localized compaction or expansion of the chain. The shape sampled by disordered chains is visualized by superposition in the principal axes frame of their inertia tensor. Ensembles of different sizes and with weighted conformers can be compared based on a similarity parameter that abstracts from the ensemble width. [ABSTRACT FROM AUTHOR]

The human Vitamin K Epoxide Reductase Complex (hVKORC1), a key enzyme transforming vitamin K into the form necessary for blood clotting, requires for its activation the reducing equivalents delivered by its redox partner through thiol-disulfide exchange reactions. The luminal loop (L-loop) is the principal mediator of hVKORC1 activation, and it is a region frequently harbouring numerous missense mutations. Four L-loop hVKORC1 mutants, suggested in vitro as either resistant (A41S, H68Y) or completely inactive (S52W, W59R), were studied in the oxidised state by numerical approaches (in silico). The DYNASOME and POCKETOME of each mutant were characterised and compared to the native protein, recently described as a modular protein composed of the structurally stable transmembrane domain (TMD) and the intrinsically disordered L-loop, exhibiting quasi-independent dynamics. The DYNASOME of mutants revealed that L-loop missense point mutations impact not only its folding and dynamics, but also those of the TMD, highlighting a strong mutation-specific interdependence between these domains. Another consequence of the mutation-induced effects manifests in the global changes (geometric, topological, and probabilistic) of the newly detected cryptic pockets and the alternation of the recognition properties of the L-loop with its redox protein. Based on our results, we postulate that (i) intra-protein allosteric regulation and (ii) the inherent allosteric regulation and cryptic pockets of each mutant depend on its DYNASOME; and (iii) the recognition of the redox protein by hVKORC1 (INTERACTOME) depend on their DYNASOME. This multifaceted description of proteins produces "omics" data sets, crucial for understanding the physiological processes of proteins and the pathologies caused by alteration of the protein properties at various "omics" levels. Additionally, such characterisation opens novel perspectives for the development of "allo-network drugs" essential for the treatment of blood disorders. [ABSTRACT FROM AUTHOR]

Nucleoplasmin (NPM) histone chaperones regulate distinct processes in the nucleus and nucleolus. While intrinsically disordered regions (IDRs) are hallmarks of NPMs, it is not clear whether all NPM functions require these unstructured features. We assessed the importance of IDRs in a yeast NPM‐like protein and found that regulation of rDNA copy number and genetic interactions with the nucleolar RNA surveillance machinery require the highly conserved FKBP prolyl isomerase domain, but not the NPM domain or IDRs. By contrast, transcriptional repression in the nucleus requires IDRs. Furthermore, multiple lysines in polyacidic serine/lysine motifs of IDRs are required for both lysine polyphosphorylation and NPM‐mediated transcriptional repression. These results demonstrate that this NPM‐like protein relies on IDRs only for some of its chromatin‐related functions. [ABSTRACT FROM AUTHOR]

The circumsporozoite protein (CSP) is the main surface antigen of the Plasmodium sporozoite (SPZ) and forms the basis of the currently only licensed anti‐malarial vaccine (RTS,S/AS01). CSP uniformly coats the SPZ and plays a pivotal role in its immunobiology, in both the insect and the vertebrate hosts. Although CSP's N‐terminal domain (CSPN) has been reported to play an important role in multiple CSP functions, a thorough biophysical and structural characterization of CSPN is currently lacking. Here, we present an alternative method for the recombinant production and purification of CSPN from Plasmodium falciparum (PfCSPN), which provides pure, high‐quality protein preparations with high yields. Through an interdisciplinary approach combining in‐solution experimental methods and in silico analyses, we provide strong evidence that PfCSPN is an intrinsically disordered region displaying some degree of compaction. [ABSTRACT FROM AUTHOR]

DNA hybridization probes are commonly used tools to discriminate clinically important single nucleotide variants (SNVs) but often work at elevated temperatures with very narrow temperature intervals (ΔT). Herein, we investigated the thermodynamic basis of the narrow ΔT both in silico and experimentally. Our study revealed that the high entropy penalty of classic hybridization probe designs was the key attributor for the narrow ΔT. Guided by this finding, we further introduced an entropy‐compensate probe (Sprobe) design by coding intrinsic disorder into a stem‐loop hybridization probe. Sprobe expanded ΔT from less than 10 °C to over 30 °C. Moreover, both ΔT and the optimal reaction temperature can be fine‐tuned by simply altering the length of the loop domain. Sprobe was clinically validated by analyzing EGFR L858R mutation in 36 pairs of clinical tumor tissue samples collected from lung cancer patients, which revealed 100 % clinical sensitivity and specificity. We anticipate that our study will serve as a general guide for designing thermal robust hybridization probes for clinical diagnostics. [ABSTRACT FROM AUTHOR]

DNA hybridization probes are commonly used tools to discriminate clinically important single nucleotide variants (SNVs) but often work at elevated temperatures with very narrow temperature intervals (ΔT). Herein, we investigated the thermodynamic basis of the narrow ΔT both in silico and experimentally. Our study revealed that the high entropy penalty of classic hybridization probe designs was the key attributor for the narrow ΔT. Guided by this finding, we further introduced an entropy‐compensate probe (Sprobe) design by coding intrinsic disorder into a stem‐loop hybridization probe. Sprobe expanded ΔT from less than 10 °C to over 30 °C. Moreover, both ΔT and the optimal reaction temperature can be fine‐tuned by simply altering the length of the loop domain. Sprobe was clinically validated by analyzing EGFR L858R mutation in 36 pairs of clinical tumor tissue samples collected from lung cancer patients, which revealed 100 % clinical sensitivity and specificity. We anticipate that our study will serve as a general guide for designing thermal robust hybridization probes for clinical diagnostics. [ABSTRACT FROM AUTHOR]

For about 30 years, SPEN has been the subject of research in many different fields due to its variety of functions and its conservation throughout a wide spectrum of species, like worms, arthropods, and vertebrates. To date, 216 orthologues have been documented. SPEN had been studied for its role in gene regulation in the context of cell signaling, including the NOTCH or nuclear hormone receptor signaling pathways. More recently, SPEN has been identified as a major regulator of initiation of chromosome-wide gene silencing during X chromosome inactivation (XCI) in mammals, where its function remains to be fully understood. Dependent on the biological context, SPEN functions via mechanisms which include different domains. While some domains of SPEN are highly conserved in sequence and secondary structure, species-to-species differences exist that might lead to mechanistic differences. Initiation of XCI appears to be different between humans and mice, which raises additional questions about the extent of generalization of SPEN's function in XCI. In this review, we dissect the mechanism of SPEN in XCI. We discuss its subregions and domains, focusing on its role as a major regulator. We further highlight species-related research, specifically of mouse and human SPEN, with the aim to reveal and clarify potential species-to-species differences in SPEN's function. [ABSTRACT FROM AUTHOR]

The evolution and diversification of proteins capable of remodeling domains has been critical for transcriptional reprogramming during cell fate determination in multicellular eukaryotes. Chromatin remodeling proteins of the CHD3 family have been shown to have important and antagonistic impacts on seed development in the model plant, Arabidopsis thaliana , yet the basis of this functional divergence remains unknown. In this study, we demonstrate that genes encoding the CHD3 proteins PICKLE (PKL) and PICKLE-RELATED 2 (PKR2) originated from a duplication event during the diversification of crown Brassicaceae, and that these homologs have undergone distinct evolutionary trajectories since this duplication, with PKR2 fast evolving under positive selection, while PKL is subject to purifying selection. We find that the rapid evolution of PKR2 under positive selection reduces the encoded protein's intrinsic disorder, possibly suggesting a tertiary structure configuration which differs from that of PKL. Our whole genome transcriptome analysis in seeds of pkr2 and pkl mutants reveals that they act antagonistically on the expression of specific sets of genes, providing a basis for their differing roles in seed development. Our results provide insights into how gene duplication and neofunctionalization can lead to differing and antagonistic selective pressures on transcriptomes during plant reproduction, as well as on the evolutionary diversification of the CHD3 family within seed plants. [ABSTRACT FROM AUTHOR]

Transcription is an essential process in biology whereby gene-specific transcription factors target sites on DNA to recruit the basal transcription machinery that will produce messenger RNA (mRNA). It is a highly regulated multi-step process that involves many proteins and protein complexes. Transcription factors, the proteins that mark genes for activation, and other transcriptional regulators are highly enriched in low-complexity disordered regions, which are strongly linked to multivalent binding and phase separation. These disordered regions can form multivalent dynamic complexes that are essential for many aspects of transcription. Many of these proteins can phase separate in vitro and show evidence of phase separation in vivo. Whether these interactions represent biologically relevant phase separation in vivo is controversial. However, what these events do demonstrate is that many transcriptional proteins co-cluster with other factors in vivo , forming multivalent dynamic clusters that contribute to transcriptional events. We review some of these recently investigated events and consider how they contribute to our understanding of transcription. Proteins that regulate transcription are highly intrinsically disordered, which is strongly linked to multivalent binding and liquid–liquid phase separation. The biological relevance of widespread phase separation is controversial, but many transcriptional proteins co-cluster with other factors, forming multiprotein–DNA complexes and multivalent dynamic hubs to target DNA and initiate transcription. Nucleic acids and other biopolymers (green, purple) and transcription factors (blue) form multivalent networks. TFs (blue) make direct interactions with DNA (orange). Multiple TFs (multiple colours) interact with DNA (orange) to initiate transcription. Dark green indicates concentrated liquid droplets surrounded by a low concentration liquid phase. [ABSTRACT FROM AUTHOR]

In recent years, it has become clear that intrinsically disordered protein segments play diverse functional roles in many cellular processes, thus leading to a reassessment of the classical structure–function paradigm. One class of intrinsically disordered protein segments is entropic clocks, corresponding to unstructured random protein chains involved in timing cellular processes. Such clocks were shown to modulate ion channel processes underlying action potential generation, propagation, and transmission. In this review, we survey the role of entropic clocks in timing intra- and inter-molecular binding events of voltage-activated potassium channels involved in gating and clustering processes, respectively, and where both are known to occur according to a similar 'ball and chain' mechanism. We begin by delineating the thermodynamic and timing signatures of a 'ball and chain'-based binding mechanism involving entropic clocks, followed by a detailed analysis of the use of such a mechanism in the prototypical Shaker voltage-activated K+ channel model protein, with particular emphasis on ion channel clustering. We demonstrate how 'chain'-level alternative splicing of the Kv channel gene modulates entropic clock-based 'ball and chain' inactivation and clustering channel functions. As such, the Kv channel model system exemplifies how linkage between alternative splicing and intrinsic disorder enables the functional diversity underlying changes in electrical signaling. [ABSTRACT FROM AUTHOR]

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]

Protein–protein interactions (PPIs) play a crucial role in numerous molecular processes. Despite many efforts, mechanisms governing molecular recognition between interacting proteins remain poorly understood and it is particularly challenging to predict from sequence whether two proteins can interact. Here we present a new method to tackle this challenge using intrinsically disordered regions (IDRs). IDRs are protein segments that are functional despite lacking a single invariant three‐dimensional structure. The prevalence of IDRs in eukaryotic proteins suggests that IDRs are critical for interactions. To test this hypothesis, we predicted PPIs using IDR sequences in candidate proteins in humans. Moreover, we divide the PPI prediction problem into two specific subproblems and adapt appropriate training and test strategies based on problem type. Our findings underline the importance of defining clearly the problem type and show that sequences encoding IDRs can aid in predicting specific features of the protein interaction network of intrinsically disordered proteins. Our findings further suggest that accounting for IDRs in future analyses should accelerate efforts to elucidate the eukaryotic PPI network. [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]

LC8, a ubiquitous and highly conserved hub protein, binds over 100 proteins involved in numerous cellular functions, including cell death, signaling, tumor suppression, and viral infection. LC8 binds intrinsically disordered proteins (IDPs), and although several of these contain multiple LC8 binding motifs, the effects of multivalency on complex formation are unclear. Drosophila ASCIZ has seven motifs that vary in sequence and inter-motif linker lengths, especially within subdomain QT2–4 containing the second, third, and fourth LC8 motifs. Using isothermal-titration calorimetry, analytical-ultracentrifugation, and native mass-spectrometry of QT2–4 variants, with methodically deactivated motifs, we show that inter-motif spacing and specific motif sequences combine to control binding affinity and compositional heterogeneity of multivalent duplexes. A short linker separating strong and weak motifs results in stable duplexes but forms off-register structures at high LC8 concentrations. Contrastingly, long linkers engender lower cooperativity and heterogeneous complexation at low LC8 concentrations. Accordingly, two-mers, rather than the expected three-mers, dominate negative-stain electron-microscopy images of QT2–4. Comparing variants containing weak-strong and strong-strong motif combinations demonstrates sequence also regulates IDP/LC8 assembly. The observed trends persist for trivalent ASCIZ subdomains: QT2–4, with long and short linkers, forms heterogeneous complexes, whereas QT4–6, with similar mid-length linkers, forms homogeneous complexes. Implications of linker length variations for function are discussed. [ABSTRACT FROM AUTHOR]

This article is a Commentary on Gómez‐Pérez et al. (2023), 239: 2320–2334. [ABSTRACT FROM AUTHOR]

One of key features of intrinsically disordered regions (IDRs) is facilitation of protein–protein and protein–nucleic acids interactions. These disordered binding regions include molecular recognition features (MoRFs), short linear motifs (SLiMs) and longer binding domains. Vast majority of current predictors of disordered binding regions target MoRFs, with a handful of methods that predict SLiMs and disordered protein-binding domains. A new and broader class of disordered binding regions, linear interacting peptides (LIPs), was introduced recently and applied in the MobiDB resource. LIPs are segments in protein sequences that undergo disorder-to-order transition upon binding to a protein or a nucleic acid, and they cover MoRFs, SLiMs and disordered protein-binding domains. Although current predictors of MoRFs and disordered protein-binding regions could be used to identify some LIPs, there are no dedicated sequence-based predictors of LIPs. To this end, we introduce CLIP, a new predictor of LIPs that utilizes robust logistic regression model to combine three complementary types of inputs: co-evolutionary information derived from multiple sequence alignments, physicochemical profiles and disorder predictions. Ablation analysis suggests that the co-evolutionary information is particularly useful for this prediction and that combining the three inputs provides substantial improvements when compared to using these inputs individually. Comparative empirical assessments using low-similarity test datasets reveal that CLIP secures area under receiver operating characteristic curve (AUC) of 0.8 and substantially improves over the results produced by the closest current tools that predict MoRFs and disordered protein-binding regions. The webserver of CLIP is freely available at http://biomine.cs.vcu.edu/servers/CLIP/ and the standalone code can be downloaded from http://yanglab.qd.sdu.edu.cn/download/CLIP/. [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]

Prions are proteinaceous particles that can propagate an alternative conformation to further copies of the same protein. They have been described in mammals, fungi, bacteria and archaea. Furthermore, across diverse organisms from bacteria to eukaryotes, prion-like proteins that have similar sequence characters are evident. Such prion-like proteins have been linked to pathomechanisms of amyotrophic lateral sclerosis (ALS) in humans, in particular TDP43, FUS, TAF15, EWSR1 and hnRNPA2. Because of the desire to study human disease-linked proteins in model organisms, and to gain insights into the functionally important parts of these proteins and how they have changed across hundreds of millions of years of evolution, we analyzed how the sequence traits of these five proteins have evolved across eukaryotes, including plants and metazoa. We discover that the RNA-binding domain architecture of these proteins is deeply conserved since their emergence. Prion-like regions are also deeply and widely conserved since the origination of the protein families for FUS, TAF15 and EWSR1, and since the last common ancestor of metazoa for TDP43 and hnRNPA2. Prion-like composition is uncommon or weak in any plant orthologs observed, however in TDP43 many plant proteins have equivalent regions rich in other amino acids (namely glycine and tyrosine and/or serine) that may be linked to stress granule recruitment. Deeply conserved low-complexity domains are identified that likely have functional significance. [ABSTRACT FROM AUTHOR]

Rabies is a neurological disease that causes between 40,000 and 70,000 deaths every year. Once a rabies patient has become symptomatic, there is no effective treatment for the illness, and in unvaccinated individuals, the case-fatality rate of rabies is close to 100%. French scientists Louis Pasteur and Émile Roux developed the first vaccine for rabies in 1885. If administered before the virus reaches the brain, the modern rabies vaccine imparts long-lasting immunity to the virus and saves more than 250,000 people every year. However, the rabies virus can suppress the host's immune response once it has entered the cells of the brain, making death likely. This study aimed to make use of disorder-based proteomics and bioinformatics to determine the potential impact that intrinsically disordered protein regions (IDPRs) in the proteome of the rabies virus might have on the infectivity and lethality of the disease. This study used the proteome of the Rabies lyssavirus (RABV) strain Pasteur Vaccins (PV), one of the best-understood strains due to its use in the first rabies vaccine, as a model. The data reported in this study are in line with the hypothesis that high levels of intrinsic disorder in the phosphoprotein (P-protein) and nucleoprotein (N-protein) allow them to participate in the creation of Negri bodies and might help this virus to suppress the antiviral immune response in the host cells. Additionally, the study suggests that there could be a link between disorder in the matrix (M) protein and the modulation of viral transcription. The disordered regions in the M-protein might have a possible role in initiating viral budding within the cell. Furthermore, we checked the prevalence of functional disorder in a set of 37 host proteins directly involved in the interaction with the RABV proteins. The hope is that these new insights will aid in the development of treatments for rabies that are effective after infection. [ABSTRACT FROM AUTHOR]

Labelling techniques such as electron paramagnetic resonance spectroscopy and single-molecule fluorescence resonance energy transfer, allow access to distances in the range of tens of angstroms, corresponding to the size of proteins and small to medium-sized protein complexes. Such measurements do not require long-range ordering and are therefore applicable to systems with partial disorder. Data from spin-label-based measurements can be processed into distance distributions that provide information about the extent of such disorder. Using such information in modelling presents several challenges, including a small number of restraints, the influence of the label itself on the measured distance and distribution width, and balancing the fitting quality of the long-range restraints with the fitting quality of other restraint subsets. Starting with general considerations about integrative and hybrid structural modelling, this review provides an overview of recent approaches to these problems and identifies where further progress is needed. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinases (CDKs) are presumed to control the cell cycle by phosphorylating a large number of proteins involved in S-phase and mitosis, two mechanistically disparate biological processes. While the traditional qualitative model of CDK-mediated cell cycle control relies on differences in inherent substrate specificity between distinct CDK-cyclin complexes, they are largely dispensable according to the opposing quantitative model, which states that changes in the overall CDK activity level promote orderly progression through S-phase and mitosis. However, a mechanistic explanation for how such an activity can simultaneously regulate many distinct proteins is lacking. New evidence suggests that the CDK-dependent phosphorylation of ostensibly very diverse proteins might be achieved due to underlying similarity of phosphorylation sites and of the biochemical effects of their phosphorylation: they are preferentially located within intrinsically disordered regions of proteins that are components of membraneless organelles, and they regulate phase separation. Here, we review this evidence and suggest a mechanism for how a single enzyme's activity can generate the dynamics required to remodel the cell at mitosis. [ABSTRACT FROM AUTHOR]

Intrinsically disordered regions (IDRs) carry out many cellular functions and vary in length and placement in protein sequences. This diversity leads to variations in the underlying compositional biases, which were demonstrated for the short vs. long IDRs. We analyze compositional biases across four classes of disorder: fully disordered proteins; short IDRs; long IDRs; and binding IDRs. We identify three distinct biases: for the fully disordered proteins, the short IDRs and the long and binding IDRs combined. We also investigate compositional bias for putative disorder produced by leading disorder predictors and find that it is similar to the bias of the native disorder. Interestingly, the accuracy of disorder predictions across different methods is correlated with the correctness of the compositional bias of their predictions highlighting the importance of the compositional bias. The predictive quality is relatively low for the disorder classes with compositional bias that is the most different from the "generic" disorder bias, while being much higher for the classes with the most similar bias. We discover that different predictors perform best across different classes of disorder. This suggests that no single predictor is universally best and motivates the development of new architectures that combine models that target specific disorder classes. [ABSTRACT FROM AUTHOR]

Much of plant development depends on cell-to-cell redistribution of the plant hormone auxin, which is facilitated by the plasma membrane (PM) localized PIN FORMED (PIN) proteins. Auxin export activity, developmental roles, subcellular trafficking, and polarity of PINs have been well studied, but their structure remains elusive besides a rough outline that they contain two groups of 5 alpha-helices connected by a large hydrophilic loop (HL). Here, we focus on the PIN1 HL as we could produce it in sufficient quantities for biochemical investigations to provide insights into its secondary structure. Circular dichroism (CD) studies revealed its nature as an intrinsically disordered protein (IDP), manifested by the increase of structure content upon thermal melting. Consistent with IDPs serving as interaction platforms, PIN1 loops homodimerize. PIN1 HL cytoplasmic overexpression in Arabidopsis disrupts early endocytic trafficking of PIN1 and PIN2 and causes defects in the cotyledon vasculature formation. In summary, we demonstrate that PIN1 HL has an intrinsically disordered nature, which must be considered to gain further structural insights. Some secondary structures may form transiently during pairing with known and yet-to-be-discovered interactors. [ABSTRACT FROM AUTHOR]

Before the SARS-CoV-2 Omicron variant emergence, shell disorder models (SDM) suggested that an attenuated precursor from pangolins may have entered humans in 2017 or earlier. This was based on a shell disorder analysis of SARS-CoV-1/2 and pangolin-Cov-2017. The SDM suggests that Omicron is attenuated with almost identical N (inner shell) disorder as pangolin-CoV-2017 (N-PID (percentage of intrinsic disorder): 44.8% vs. 44.9%—lower than other variants). The outer shell disorder (M-PID) of Omicron is lower than that of other variants and pangolin-CoV-2017 (5.4% vs. 5.9%). COVID-19-related CoVs have the lowest M-PIDs (hardest outer shell) among all CoVs. This is likely to be responsible for the higher contagiousness of SARS-CoV-2 and Omicron, since hard outer shell protects the virion from salivary/mucosal antimicrobial enzymes. Phylogenetic study using M reveals that Omicron branched off from an ancestor of the Wuhan-Hu-1 strain closely related to pangolin-CoVs. M, being evolutionarily conserved in COVID-19, is most ideal for COVID-19 phylogenetic study. Omicron may have been hiding among burrowing animals (e.g., pangolins) that provide optimal evolutionary environments for attenuation and increase shell hardness, which is essential for fecal–oral–respiratory transmission via buried feces. Incoming data support SDM e.g., the presence of fewer infectious particles in the lungs than in the bronchi upon infection. [ABSTRACT FROM AUTHOR]

The early stages of protein misfolding and aggregation involve disordered and partially folded protein conformers that contain a high degree of dynamic disorder. These dynamic species may undergo large-scale intra-molecular motions of intrinsically disordered protein (IDP) precursors, or flexible, low affinity inter-molecular binding in oligomeric assemblies. In both cases, generating atomic level visualization of the interconverting species that captures the conformations explored and their physico-chemical properties remains hugely challenging. How specific sub-ensembles of conformers that are on-pathway to aggregation into amyloid can be identified from their aggregation-resilient counterparts within these large heterogenous pools of rapidly moving molecules represents an additional level of complexity. Here, we describe current experimental and computational approaches designed to capture the dynamic nature of the early stages of protein misfolding and aggregation, and discuss potential challenges in describing these species because of the ensemble averaging of experimental restraints that arise from motions on the millisecond timescale. We give a perspective of how machine learning methods can be used to extract aggregation-relevant sub-ensembles and provide two examples of such an approach in which specific interactions of defined species within the dynamic ensembles of α-synuclein (αSyn) and β2-microgloblulin (β2m) can be captured and investigated. [ABSTRACT FROM AUTHOR]

Proteins with intrinsically disordered regions (IDRs) are common among eukaryotes. Many IDRs interact with nucleic acids and proteins. Annotation of these interactions is supported by computational predictors, but to date, only one tool that predicts interactions with nucleic acids was released, and recent assessments demonstrate that current predictors offer modest levels of accuracy. We have developed DeepDISOBind, an innovative deep multi-task architecture that accurately predicts deoxyribonucleic acid (DNA)-, ribonucleic acid (RNA)- and protein-binding IDRs from protein sequences. DeepDISOBind relies on an information-rich sequence profile that is processed by an innovative multi-task deep neural network, where subsequent layers are gradually specialized to predict interactions with specific partner types. The common input layer links to a layer that differentiates protein- and nucleic acid-binding, which further links to layers that discriminate between DNA and RNA interactions. Empirical tests show that this multi-task design provides statistically significant gains in predictive quality across the three partner types when compared to a single-task design and a representative selection of the existing methods that cover both disorder- and structure-trained tools. Analysis of the predictions on the human proteome reveals that DeepDISOBind predictions can be encoded into protein-level propensities that accurately predict DNA- and RNA-binding proteins and protein hubs. DeepDISOBind is available at https://www.csuligroup.com/DeepDISOBind/ [ABSTRACT FROM AUTHOR]

Intrinsic disorder prediction field develops, assesses, and deploys computational predictors of disorder in protein sequences and constructs and disseminates databases of these predictions. Over 40 years of research resulted in the release of numerous resources. We identify and briefly summarize the most comprehensive to date collection of over 100 disorder predictors. We focus on their predictive models, availability and predictive performance. We categorize and study them from a historical point of view to highlight informative trends. We find a consistent trend of improvements in predictive quality as newer and more advanced predictors are developed. The original focus on machine learning methods has shifted to meta-predictors in early 2010s, followed by a recent transition to deep learning. The use of deep learners will continue in foreseeable future given recent and convincing success of these methods. Moreover, a broad range of resources that facilitate convenient collection of accurate disorder predictions is available to users. They include web servers and standalone programs for disorder prediction, servers that combine prediction of disorder and disorder functions, and large databases of pre-computed predictions. We also point to the need to address the shortage of accurate methods that predict disordered binding regions. [ABSTRACT FROM AUTHOR]

Compositionally-biased (CB) regions in biological sequences are enriched for a subset of sequence residue types. These can be shorter regions with a concentrated bias (i.e., those termed 'low-complexity'), or longer regions that have a compositional skew. These regions comprise a prominent class of the uncharacterized 'dark matter' of the protein universe. Here, I report the latest version of the fLPS package for the annotation of CB regions, which includes added consideration of DNA sequences, to label the eight possible biased regions of DNA. In this version, the user is now able to restrict analysis to a specified subset of residue types, and also to filter for previously annotated domains to enable detection of discontinuous CB regions. A 'thorough' option has been added which enables the labelling of subtler biases, typically made from a skew for several residue types. In the output, protein CB regions are now labelled with bias classes reflecting the physico-chemical character of the biasing residues. The fLPS 2.0 package is available from: https://github.com/pmharrison/flps2 or in a Supplemental File of this paper. [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]

Intrinsically disordered proteins (IDPs) interact with globular proteins through a variety of mechanisms, resulting in the structurally heterogeneous ensembles known as fuzzy complexes. While there exists a reasonable comprehension on how IDP sequence determines the unbound IDP ensemble, little is known about what shapes the structural characteristics of IDPs bound to their targets. Using a statistical thermodynamic model, we show that the targetbound ensembles are determined by a simple code that combines the IDP sequence and the distribution of IDP-target interaction hotspots. These two parameters define the conformational space of target-bound IDPs and rationalize the observed structural heterogeneity of fuzzy complexes. The presented model successfully reproduces the dynamical signatures of target-bound IDPs from the NMR relaxation experiments as well as the changes of interaction affinity and the IDP helicity induced by mutations. The model explains how the target-bound IDP ensemble adapts to mutations in order to achieve an optimal balance between conformational freedom and interaction energy. Taken together, the presented sequence-ensemble relationship of fuzzy complexes explains the different manifestations of IDP disorder in folding-upon-binding processes. [ABSTRACT FROM AUTHOR]

The known polyspecificity of antibodies, which is crucial for efficient immune response, is determined by the conformational flexibility and intrinsic disorder encoded in local peculiarities of the amino acid sequence of antibodies within or in the vicinity of their complementarity determining regions. Similarly, epitopes represent fuzzy binding sites, which are also characterized by local structural flexibility. Existing data suggest that the efficient interactions between antigens and antibodies rely on the conformational mobility and some disorder of their binding sites and therefore can be relatively well described by the "flexible lock – adjustable key" model, whereas both, extreme order (rigid lock-and-key) and extreme disorder (viral shape-shifters) are not compatible with the efficient antigen-antibody interactions and are not present in immune interactions. [ABSTRACT FROM AUTHOR]

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL's α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including β2, β3-α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3-α3 and progress to β5 and β4-α4, ultimately leading to the irreversible unfolding of regions that form LPL's catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL's α/β-hydrolase domain (Tm of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (Tm of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ~20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism. [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]

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]

Phosphorylation sites are hyperabundant in the eukaryotic disordered proteome, suggesting that conformational fluctuations play a major role in determining to what extent a kinase interacts with a particular substrate. In biophysical terms, substrate selectivity may be determined not just by the structural–chemical complementarity between the kinase and its protein substrates but also by the free energy difference between the conformational ensembles that are, or are not, recognized by the kinase. To test this hypothesis, we developed a statistical-thermodynamics-based informatics framework, which allows us to probe for the contribution of equilibrium fluctuations to phosphorylation, as evaluated by the ability to predict Ser/Thr/Tyr phosphorylation sites in the disordered proteome. Essential to this framework is a decomposition of substrate sequence information into two types: vertical information encoding conserved kinase specificity motifs and horizontal information encoding substrate conformational equilibrium that is embedded, but often not apparent, within position-specific conservation patterns. We find not only that conformational fluctuations play a major role but also that they are the dominant contribution to substrate selectivity. In fact, the main substrate classifier distinguishing selectivity is the magnitude of change in local compaction of the disordered chain upon phosphorylation of these mostly singly phosphorylated sites. In addition to providing fundamental insights into the consequences of phosphorylation across the proteome, our approach provides a statistical-thermodynamic strategy for partitioning any sequencebased search into contributions from structural–chemical complementarity and those from changes in conformational equilibrium. [ABSTRACT FROM AUTHOR]

Biominerals are crucial to the fitness of many organism and studies of the mechanisms of biomineralization are driving research into novel materials. Biomineralization is generally controlled by a matrix of organic molecules including proteins, so proteomic studies of biominerals are important for understanding biomineralization mechanisms. Many such studies identify large numbers of proteins of unknown function, which are often of low sequence complexity and biased in their amino acid composition. A lack of user-friendly tools to find patterns in such sequences and robustly analyse their statistical properties relative to the background proteome means that they are often neglected in follow-up studies. Here we present ProminTools, a user-friendly package for comparison of two sets of protein sequences in terms of their global properties and motif content. Outputs include data tables, graphical summaries in an html file and an R-script as a starting point for data-set specific visualizations. We demonstrate the utility of ProminTools using a previously published shell matrix proteome of the giant limpet Lottia gigantea. [ABSTRACT FROM AUTHOR]

Experimental annotations of intrinsic disorder are available for 0.1% of 147 000 000 of currently sequenced proteins. Over 60 sequence-based disorder predictors were developed to help bridge this gap. Current benchmarks of these methods assess predictive performance on datasets of proteins; however, predictions are often interpreted for individual proteins. We demonstrate that the protein-level predictive performance varies substantially from the dataset-level benchmarks. Thus, we perform first-of-its-kind protein-level assessment for 13 popular disorder predictors using 6200 disorder-annotated proteins. We show that the protein-level distributions are substantially skewed toward high predictive quality while having long tails of poor predictions. Consequently, between 57% and 75% proteins secure higher predictive performance than the currently used dataset-level assessment suggests, but as many as 30% of proteins that are located in the long tails suffer low predictive performance. These proteins typically have relatively high amounts of disorder, in contrast to the mostly structured proteins that are predicted accurately by all 13 methods. Interestingly, each predictor provides the most accurate results for some number of proteins, while the best-performing at the dataset-level method is in fact the best for only about 30% of proteins. Moreover, the majority of proteins are predicted more accurately than the dataset-level performance of the most accurate tool by at least four disorder predictors. While these results suggests that disorder predictors outperform their current benchmark performance for the majority of proteins and that they complement each other, novel tools that accurately identify the hard-to-predict proteins and that make accurate predictions for these proteins are needed. [ABSTRACT FROM AUTHOR]

Cooperativity enhances the responsiveness of biomolecular receptors to small changes in the concentration of their target ligand, albeit with a concomitant reduction in affinity. The binding midpoint of a two-site receptor with a Hill coefficient of 1.9, for example, must be at least 19 times higher than the dissociation constant of the higher affinity of its two binding sites. This trade-off can be overcome, however, by the extra binding energy provided by the addition of more binding sites, which can be used to achieve highly cooperative receptors that still retain high affinity. Exploring this experimentally, we have employed an “intrinsic disorder” mechanism to design two cooperative, three-binding-site receptors starting from a single-site—and thus noncooperative—doxorubicin-binding aptamer. The first receptor follows a binding energy landscape that partitions the energy provided by the additional binding event to favor affinity, achieving a Hill coefficient of 1.9 but affinity within a factor of 2 of the parent aptamer. The binding energy landscape of the second receptor, in contrast, partitions more of this energy toward cooperativity, achieving a Hill coefficient of 2.3, but at the cost of 4-fold poorer affinity than that of the parent aptamer. The switch between these two behaviors is driven primarily by the affinity of the receptors’ second binding event, which serves as an allosteric “gatekeeper” defining the extent to which the system is weighted toward higher cooperativity or higher affinity. [ABSTRACT FROM AUTHOR]

Prions are self-propagating alternative states of protein domains. They are linked to both diseases and functional protein roles in eukaryotes. Prion-forming domains in Saccharomyces cerevisiae are typically domains with high intrinsic protein disorder (i.e., that remain unfolded in the cell during at least some part of their functioning), that are converted to self-replicating amyloid forms. S. cerevisiae is a member of the fungal class Saccharomycetes, during the evolution of which a large population of prion-like domains has appeared. It is still unclear what principles might govern the molecular evolution of prion-forming domains, and intrinsically disordered domains generally. Here, it is discovered that in a set of such prion-forming domains some evolve in the fungal class Saccharomycetes in such a way as to absorb general mutation biases across millions of years, whereas others do not, indicating a spectrum of selection pressures on composition and sequence. Thus, if the bias-absorbing prion formers are conserving a prion-forming capability, then this capability is not interfered with by the absorption of bias changes over the duration of evolutionary epochs. Evidence is discovered for selective constraint against the occurrence of lysine residues (which likely disrupt prion formation) in S. cerevisiae prion-forming domains as they evolve across Saccharomycetes. These results provide a case study of the absorption of mutational trends by compositionally biased domains, and suggest methodology for assessing selection pressures on the composition of intrinsically disordered regions. [ABSTRACT FROM AUTHOR]

Pathogen infection triggers extensive reprogramming of the plant transcriptome, including numerous genes the function of which is unknown. Due to their wide taxonomic distribution, genes encoding proteins with Domains of Unknown Function (DUFs) activated upon pathogen challenge likely play important roles in disease. In Arabidopsis thaliana , we identified thirteen genes harboring a DUF4228 domain in the top 10% most induced genes after infection by the fungal pathogen Sclerotinia sclerotiorum. Based on functional information collected through homology and contextual searches, we propose to refer to this domain as the pathogen and abiotic stress response, cadmium tolerance, disordered region-containing (PADRE) domain. Genome-wide and phylogenetic analyses indicated that PADRE is specific to plants and diversified into 10 subfamilies early in the evolution of Angiosperms. PADRE typically occurs in small single-domain proteins with a bipartite architecture. PADRE N-terminus harbors conserved sequence motifs, while its C-terminus includes an intrinsically disordered region with multiple phosphorylation sites. A pangenomic survey of PADRE genes expression upon S. sclerotiorum inoculation in Arabidopsis , castor bean, and tomato indicated consistent expression across species within phylogenetic groups. Multi-stress expression profiling and co-expression network analyses associated AtPADRE genes with the induction of anthocyanin biosynthesis and responses to chitin and to hypoxia. Our analyses reveal patterns of sequence and expression diversification consistent with the evolution of a role in disease resistance for an uncharacterized family of plant genes. These findings highlight PADRE genes as prime candidates for the functional dissection of mechanisms underlying plant disease resistance to fungi. [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]

Pub1 protein is an important RNA-binding protein functional in stress granule assembly in budding yeast Saccharomyces cerevisiae and, as its co-ortholog Tia1, in humans. It is unique among proteins in evidencing prion-like aggregation in both its yeast and human forms. Previously, we noted that Pub1/Tia1 was the only protein linked to human disease that has prion-like character and and has demonstrated such aggregation in both species. Thus, we were motivated to probe further into the evolution of the Pub1/Tia1 family (and its close relative Nam8 and its orthologs) to gain a picture of how such a protein has evolved over deep evolutionary time since the last common ancestor of eukaryotes. Here, we discover that the prion-like composition of this protein family is deeply conserved across eukaryotes, as is the prion-like composition of its close relative Nam8/Ngr1. A sizeable minority of protein orthologs have multiple prion-like domains within their sequences (6–20% depending on criteria). The number of RNA-binding RRM domains is conserved at three copies over >86% of the Pub1 family (>71% of the Nam8 family), but proteins with just one or two RRM domains occur frequently in some clades, indicating that these are not due to annotation errors. Overall, our results indicate that a basic scaffold comprising three RNA-binding domains and at least one prion-like region has been largely conserved since the last common ancestor of eukaryotes, providing further evidence that prion-like aggregation may be a very ancient and conserved phenomenon for certain specific proteins. [ABSTRACT FROM AUTHOR]

Protein tandem repeats (TRs) are often associated with immunity-related functions and diseases. Since that last census of protein TRs in 1999, the number of curated proteins increased more than seven-fold and new TR prediction methods were published. TRs appear to be enriched with intrinsic disorder and vice versa. The significance and the biological reasons for this association are unknown. Here, we characterize protein TRs across all kingdoms of life and their overlap with intrinsic disorder in unprecedented detail. Using state-of-the-art prediction methods, we estimate that 50.9% of proteins contain at least one TR, often located at the sequence flanks. Positive linear correlation between the proportion of TRs and the protein length was observed universally, with Eukaryotes in general having more TRs, but when the difference in length is taken into account the difference is quite small. TRs were enriched with disorder-promoting amino acids and were inside intrinsically disordered regions. Many such TRs were homorepeats. Our results support that TRs mostly originate by duplication and are involved in essential functions such as transcription processes, structural organization, electron transport and iron-binding. In viruses, TRs are found in proteins essential for virulence. [ABSTRACT FROM AUTHOR]