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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.

Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences of glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing data base of experimentally verified IDPs (DisProt) and show that the glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using a model acidic IDP from yeast and three Glu/Asp variants, we find that while Glu/Asp support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity where glutamates can support increased helicity important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in e.g., transactivation domains and ion-binding.

With the increased focus on intrinsically disordered proteins (IDPs) and their large interactomes, the question about their specificity — or more so on their multispecificity — arise. Here we recapitulate how specificity and multispecificity are quantified and address through examples if IDPs in this respect differ from globular proteins. The conclusion is that quantitatively, globular proteins and IDPs are similar when it comes to specificity. However, compared with globular proteins, IDPs have larger interactome sizes, a phenomenon that is further enabled by their flexibility, repetitive binding motifs and propensity to adapt to different binding partners. For IDPs, this adaptability, interactome size and a higher degree of multivalency opens for new interaction mechanisms such as facilitated exchange through trimer formation and ultra-sensitivity via threshold effects and ensemble redistribution. IDPs and their interactions, thus, do not compromise the definition of specificity. Instead, it is the sheer size of their interactomes that complicates its calculation. More importantly, it is this size that challenges how we conceptually envision, interpret and speak about their specificity.

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.

Traditionally, our understanding of how proteins operate and how evolution shapes them is based on two main data sources: the overall protein fold and the protein amino acid sequence. However, a significant part of the proteome shows highly dynamic and/or structurally ambiguous behavior, which cannot be correctly represented by the traditional fixed set of static coordinates. Representing such protein behaviors remains challenging and necessarily involves a complex interpretation of conformational states, including probabilistic descriptions. Relating protein dynamics and multiple conformations to their function as well as their physiological context (e.g., post-translational modifications and subcellular localization), therefore, remains elusive for much of the proteome, with studies to investigate the effect of protein dynamics relying heavily on computational models. We here investigate the possibility of delineating three classes of protein conformational behavior: order, disorder, and ambiguity. These definitions are explored based on three different datasets, using interpretable machine learning from a set of features, from AlphaFold2 to sequence-based predictions, to understand the overlap and differences between these datasets. This forms the basis for a discussion on the current limitations in describing the behavior of dynamic and ambiguous proteins.

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.

The enzymatic, asymmetric reduction of imines is catalyzed by imine reductases (IREDs), members of the short‐chain dehydrogenase/reductase (SDR) family, and β‐hydroxy acid dehydrogenase (βHAD) variants. Systematic evaluation of the structures and substrate‐binding sites of the three enzyme families has revealed four common principles for imine reduction: structurally conserved cofactor‐binding domains; tyrosine, aspartate, or glutamate as proton donor; at least four characteristic flanking residues that adapt the donor's pK a and polarize the substrate; and a negative electrostatic potential in the substrate‐binding site to stabilize the transition state. As additional catalytically relevant positions, we propose alternative proton donors in IREDs and βHADs as well as proton relays in IREDs, βHADs, and SDRs. The functional role of flanking residues was experimentally confirmed by alanine scanning of the imine‐reducing SDR from Zephyranthes treatiae. Mutating the “gatekeeping” phenylalanine at standard position 200 resulted in a tenfold increase in imine‐reducing activity., The enzymatic, asymmetric reduction of imines to chiral amines is a valuable biocatalytic approach owing to the high enantioselectivity of the reductases. Imine reductases, short‐chain dehydrogenases/reductases, and β‐hydroxy acid dehydrogenases are known to catalyze this reaction. Our systematic evaluation of the structures and substrate‐binding sites of the three enzyme families has revealed four common principles for enzymatic imine reduction.

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. Electronic supplementary material The online version of this article (10.1007/s00239-020-09939-z) contains supplementary material, which is available to authorized users.

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., Biomolecules, 12 (10), ISSN:2218-273X

Intrinsically disordered or unstructured proteins (or regions in proteins) have been found to be important in a wide range of biological functions and implicated in many diseases. Due to the high cost and low efficiency of experimental determination of intrinsic disorder and the exponential increase of unannotated protein sequences, developing complementary computational prediction methods has been an active area of research for several decades. Here, we employed an ensemble of deep Squeeze-and-Excitation residual inception and long short-term memory (LSTM) networks for predicting protein intrinsic disorder with input from evolutionary information and predicted one-dimensional structural properties. The method, called SPOT-Disorder2, offers substantial and consistent improvement not only over our previous technique based on LSTM networks alone, but also over other state-of-the-art techniques in three independent tests with different ratios of disordered to ordered amino acid residues, and for sequences with either rich or limited evolutionary information. More importantly, semi-disordered regions predicted in SPOT-Disorder2 are more accurate in identifying molecular recognition features (MoRFs) than methods directly designed for MoRFs prediction. SPOT-Disorder2 is available as a web server and as a standalone program at https://sparks-lab.org/server/spot-disorder2/.

ErbBs are receptor tyrosine kinases involved not only in development, but also in a wide variety of diseases, particularly cancer. Their extracellular, transmembrane, juxtamembrane, and kinase folded domains were described extensively over the past 20 years, structurally and functionally. However, their whole C-terminal tails (CTs) following the kinase domain were only described at atomic resolution in the last 4 years. They were shown to be intrinsically disordered. The CTs are known to be tyrosine-phosphorylated when the activated homo- or hetero-dimers of ErbBs are formed. Their phosphorylation triggers interaction with phosphotyrosine binding (PTB) or Src Homology 2 (SH2) domains and activates several signaling pathways controling cellular motility, proliferation, adhesion, and apoptosis. Beyond this passive role of phosphorylated domain and site display for partners, recent structural and function studies unveiled active roles in regulation of phosphorylation and interaction: the CT regulates activity of the kinase domain; different phosphorylation states have different compaction levels, potentially modulating the succession of phosphorylation events; and prolines have an important role in structure, dynamics, and possibly regulatory interactions. Here, we review both the canonical role of the disordered CT domains of ErbBs as phosphotyrosine display domains and the recent findings that expand the known range of their regulation functions linked to specific structural and dynamic features.

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.

Calmodulin (CaM) is a calcium-binding protein that transduces signals to downstream proteins through target binding upon calcium binding in a time-dependent manner. Understanding the target binding process that tunes CaM’s affinity for the calcium ions (Ca2+), or vice versa, may provide insight into how Ca2+-CaM selects its target binding proteins. However, modeling of Ca2+-CaM in molecular simulations is challenging because of the gross structural changes in its central linker regions while the two lobes are relatively rigid due to tight binding of the Ca2+ to the calcium-binding loops where the loop forms a pentagonal bipyramidal coordination geometry with Ca2+. This feature that underlies the reciprocal relation between Ca2+ binding and target binding of CaM, however, has yet to be considered in the structural modeling. Here, we presented a coarse-grained model based on the Associative memory, Water mediated, Structure, and Energy Model (AWSEM) protein force field, to investigate the salient features of CaM. Particularly, we optimized the force field of CaM and that of Ca2+ ions by using its coordination chemistry in the calcium-binding loops to match with experimental observations. We presented a “community model” of CaM that is capable of sampling various conformations of CaM, incorporating various calcium-binding states, and carrying the memory of binding with various targets, which sets the foundation of the reciprocal relation of target binding and Ca2+ binding in future studies.

Intrinsically disordered proteins (IDPs) regularly constitute components of larger protein assemblies contributing to architectural stability. Two small, highly acidic IDPs have been linked to the so-called PCI complexes carrying PCI-domain subunits, including the proteasome lid and the COP9 signalosome. These two IDPs, DSS1 and CSNAP, have been proposed to have similar structural propensities and functions, but they display differences in their interactions and interactome sizes. Here we characterized the structural properties of human DSS1 and CSNAP at the residue level using NMR spectroscopy and probed their propensities to bind ubiquitin. We find that distinct structural features present in DSS1 are completely absent in CSNAP, and vice versa, with lack of relevant ubiquitin binding to CSNAP, suggesting the two proteins to have diverged in both structure and function. Our work additionally highlights that different local features of seemingly similar IDPs, even subtle sequence variance, may endow them with different functional traits. Such traits may underlie their potential to engage in multiple interactions thereby impacting their interactome sizes. This article is protected by copyright. All rights reserved.

The Wnt signalling pathway plays an important role in cell proliferation, differentiation, and fate decisions in embryonic development and the maintenance of adult tissues. The twelve armadillo (ARM) repeat-containing protein β-catenin acts as the signal transducer in this pathway. Here, we investigated the interaction between β-catenin and the intrinsically disordered transcription factor TCF7L2, comprising a very long nanomolar-affinity interface of approximately 4800 Å2 that spans ten of the twelve ARM repeats of β-catenin. First, a fluorescence reporter system for the interaction was engineered and used to determine the kinetic rate constants for the association and dissociation. The association kinetics of TCF7L2 and β-catenin were monophasic and rapid (7.3 ± 0.1 × 107 M−1·s−1), whereas dissociation was biphasic and slow (5.7 ± 0.4 × 10−4 s−1, 15.2 ± 2.8 × 10−4 s−1). This reporter system was then combined with site-directed mutagenesis to investigate the striking variability in the conformation adopted by TCF7L2 in the three different crystal structures of the TCF7L2–β-catenin complex. We found that the mutation had very little effect on the association kinetics, indicating that most interactions form after the rate-limiting barrier for association. Mutations of the N- and C-terminal subdomains of TCF7L2 that adopt relatively fixed conformations in the crystal structures had large effects on the dissociation kinetics, whereas the mutation of the labile sub-domain connecting them had negligible effect. These results point to a two-site avidity mechanism of binding with the linker region forming a “fuzzy” complex involving transient contacts that are not site-specific. Strikingly, the two mutations in the N-terminal subdomain that had the largest effects on the dissociation kinetics showed two additional phases, indicating partial flux through an alternative dissociation pathway that is inaccessible to the wild type. The results presented here provide insights into the kinetics of the molecular recognition of a long intrinsically disordered region with an elongated repeat-protein surface, a process found to involve parallel routes with sequential steps in each.

Intrinsic disorder is a natural feature of polypeptide chains, resulting in the lack of a defined three-dimensional structure. Conformational changes in intrinsically disordered regions of a protein lead to unstable β-sheet enriched intermediates, which are stabilized by intermolecular interactions with other β-sheet enriched molecules, producing stable proteinaceous aggregates. Upon misfolding, several pathways may be undertaken depending on the composition of the amino acidic string and the surrounding environment, leading to different structures. Accumulating evidence is suggesting that the conformational state of a protein may initiate signalling pathways involved both in pathology and physiology. In this review, we will summarize the heterogeneity of structures that are produced from intrinsically disordered protein domains and highlight the routes that lead to the formation of physiological liquid droplets as well as pathogenic aggregates. The most common proteins found in aggregates in neurodegenerative diseases and their structural variability will be addressed. We will further evaluate the clinical relevance and future applications of the study of the structural heterogeneity of protein aggregates, which may aid the understanding of the phenotypic diversity observed in neurodegenerative disorders.

The formation of a mature myelin sheath in the vertebrate nervous system requires specific protein-membrane interactions. Several myelin-specific proteins are involved in stacking lipid membranes into multilayered structures around axons, and misregulation of these processes may contribute to chronic demyelinating diseases. Two key proteins in myelin membrane binding and stacking are the myelin basic protein (MBP) and protein zero (P0). Other factors, including Ca 2+ , are important for the regulation of myelination. We studied the effects of ionic strength and Ca 2+ on the membrane interactions of MBP and the cytoplasmic domain of P0 (P0ct). MBP and P0ct bound and aggregated negatively charged lipid vesicles, while simultaneously folding, and both ionic strength and calcium had systematic effects on these interactions. When decreasing membrane net negative charge, the level and kinetics of vesicle aggregation were affected by both salt and Ca 2+ . The effects on lipid membrane surfaces by ions can directly affect myelin protein-membrane interactions, in addition to signalling effects in myelinating glia.

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.

Prevention of amyloid β peptide (Aβ) deposition via facilitation of Aβ binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer’s disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, to improve the affinity of this protein to monomeric Aβ by a factor of 3 (BBRC, 510(2), 248–253). Using plasmon resonance spectroscopy, we show here that another HSA ligand related to AD pathogenesis, serotonin (SRO), increases the affinity of the Aβ monomer to HSA by a factor of 7/17 for Aβ40/Aβ42, respectively. Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA’s affinity to monomeric Aβ, despite slowdown of the association and dissociation processes. Crosslinking with glutaraldehyde and dynamic light scattering experiments reveal that, compared with the TRP-induced effects, SRO binding causes more marked changes in the quaternary structure of HSA. Furthermore, molecular docking reveals distinct structural differences between SRO/TRP complexes with HSA. The disintegration of the serotonergic system during AD pathogenesis may contribute to Aβ release from HSA in the central nervous system due to impairment of the SRO-mediated Aβ trapping by HSA.

An understanding of the forces shaping protein conservation is key, both for the fundamental knowledge it represents and to allow for optimal use of evolutionary information in practical applications. Sequence conservation is typically examined at one of two levels. The first is a residue-level, where intra-protein differences are analyzed and the second is a protein-level, where inter-protein differences are studied. At a residue level, we know that solvent-accessibility is a prime determinant of conservation. By inverting this logic, we inferred that disordered regions are slightly more solvent-accessible on average than the most exposed surface residues in domains. By integrating abundance information with evolutionary data within and across proteins, we confirmed a previously reported strong surface-core association in the evolution of structured regions, but we found a comparatively weak association between disordered and structured regions. The facts that disordered and structured regions experience different structural constraints and evolve independently provide a unique setup to examine an outstanding question: why is a protein’s abundance the main determinant of its sequence conservation? Indeed, any structural or biophysical property linked to the abundance-conservation relationship should increase the relative conservation of regions concerned with that property (e.g., disordered residues with mis-interactions, domain residues with misfolding). Surprisingly, however, we found the conservation of disordered and structured regions to increase in equal proportion with abundance. This observation implies that either abundance-related constraints are structure-independent, or multiple constraints apply to different regions and perfectly balance each other.

International audience; The ASR protein family has been discovered thirty years ago in many plant species and is involved in the tolerance of various abiotic stresses such as dehydration, salinity and heat. Despite its importance, nothing is known about the conserved ABA-Water Deficit Stress Domain (ABA-WDS) of the ASR gene family. In this study, we characterized two ABA-WDS domains, isolated from durum wheat (TtABA-WDS) and barley (HvABA-WDS). Bioinformatics analysis shows that they are both consistently predicted to be intrinsically disordered. Hydrodynamic and circular dichroism analysis indicate that both domains are largely disordered but belong to different structural classes, with HvABA-WDS and TtABA-WDS adopting a PreMolten Globule-like (PMG-like) and a Random Coil-like (RC-like) conformation, respectively. In the presence of the secondary structure stabilizer trifluoroethanol (TFE) or of increasing glycerol concentrations, which mimics dehydration, the two domains acquire an α-helical structure. Interestingly, both domains are able to prevent heat- and dehydration-induced inactivation of the enzyme lactate dehydrogenase (LDH). Furthermore, heterologous expression of TtABA-WDS and HvABA-WDS in the yeast Saccharomyces cerevisiae improves its tolerance to salt, heat and cold stresses. Taken together our results converge to show that the ABA-WDS domain is an intrinsically disordered functional domain whose conformational plasticity could be instrumental to support the versatile functions attributed to the ASR family, including its role in abiotic stress tolerance. Finally, and after validation in the plant system, this domain could be used to improve crop tolerance to abiotic stresses..This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

The current Coronavirus Disease 19 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) shows similar pathology to MERS and SARS-CoV, with a current estimated fatality rate of 1.4%. Open reading frame 10 (ORF10) is a unique SARS-CoV-2 accessory protein, which contains eleven cytotoxic T lymphocyte (CTL) epitopes each of nine amino acids in length. Twenty-two unique SARS-CoV-2 ORF10 variants have been identified based on missense mutations found in sequence databases. Some of these mutations are predicted to decrease the stability of ORF10 in silico physicochemical and structural comparative analyses were carried out on SARS-CoV-2 and Pangolin-CoV ORF10 proteins, which share 97.37% amino acid (aa) homology. Though there is a high degree of ORF10 protein similarity of SARS-CoV-2 and Pangolin-CoV, there are differences of these two ORF10 proteins related to their sub-structure (loop/coil region), solubility, antigenicity and shift from strand to coil at aa position 26 (tyrosine). SARS-CoV-2 ORF10, which is apparently expressed in vivo since reactive T cell clones are found in convalescent patients should be monitored for changes which could correlate with the pathogenesis of COVID-19.

Ubiquitin is a small protein at the heart of many cellular processes, and several different protein domains are known to recognize and bind ubiquitin. A common motif for interaction with ubiquitin is the Ubiquitin Interacting Motif (UIM), characterized by a conserved sequence signature and often found in multi-domain proteins. Multi-domain proteins with intrinsically disordered regions mediate interactions with multiple partners, orchestrating diverse pathways. Short linear motifs for binding are often embedded in these disordered regions and play crucial roles in modulating protein function. In this work, we investigated the structural propensities of UIMs using molecular dynamics simulations and NMR chemical shifts. Despite the structural portrait depicted by X-crystallography of stable helical structures, we show that UIMs feature both helical and intrinsically disordered conformations. Our results shed light on a new class of disordered UIMs. This group is here exemplified by the C-terminal domain of one isoform of ataxin-3 and a group of ubiquitin-specific proteases. Intriguingly, UIMs not only bind ubiquitin. They can be a recruitment point for other interactors, such as parkin and the heat shock protein Hsc70-4. Disordered UIMs can provide versatility and new functions to the client proteins, opening new directions for research on their interactome.

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

A large fraction of peptides or protein regions are disordered in isolation and fold upon binding. These regions, also called MoRFs, SLiMs or LIPs, are often associated with signaling and regulation processes. However, despite their importance, only a limited number of examples are available in public databases and their automatic detection at the proteome level is problematic. Here we present FLIPPER, an automatic method for the detection of structurally linear sub-regions or peptides that interact with another chain in a protein complex. FLIPPER is a random forest classification that takes the protein structure as input and provides the propensity of each amino acid to be part of a LIP region. Models are built taking into consideration structural features such as intra- and inter-chain contacts, secondary structure, solvent accessibility in both bound and unbound state, structural linearity and chain length. FLIPPER is accurate when evaluated on non-redundant independent datasets, 99% precision and 99% sensitivity on PixelDB-25 and 87% precision and 88% sensitivity on DIBS-25. Finally, we used FLIPPER to process the entire Protein Data Bank and identified different classes of LIPs based on different binding modes and partner molecules. We provide a detailed description of these LIP categories and show that a large fraction of these regions are not detected by disorder predictors. All FLIPPER predictions are integrated in the MobiDB 4.0 database.

With over 60 disorder predictors, users need help navigating the predictor selection task. We review 28 surveys of disorder predictors, showing that only 11 include assessment of predictive performance. We identify and address a few drawbacks of these past surveys. To this end, we release a novel benchmark dataset with reduced similarity to the training sets of the considered predictors. We use this dataset to perform a first-of-its-kind comparative analysis that targets two large functional families of disordered proteins that interact with proteins and with nucleic acids. We show that limiting sequence similarity between the benchmark and the training datasets has a substantial impact on predictive performance. We also demonstrate that predictive quality is sensitive to the use of the well-annotated order and inclusion of the fully structured proteins in the benchmark datasets, both of which should be considered in future assessments. We identify three predictors that provide favorable results using the new benchmark set. While we find that VSL2B offers the most accurate and robust results overall, ESpritz-DisProt and SPOT-Disorder perform particularly well for disordered proteins. Moreover, we find that predictions for the disordered protein-binding proteins suffer low predictive quality compared to generic disordered proteins and the disordered nucleic acids-binding proteins. This can be explained by the high disorder content of the disordered protein-binding proteins, which makes it difficult for the current methods to accurately identify ordered regions in these proteins. This finding motivates the development of a new generation of methods that would target these difficult-to-predict disordered proteins. We also discuss resources that support users in collecting and identifying high-quality disorder predictions.

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.

The proteome of the malaria parasite Plasmodium falciparum is notable for the pervasive occurrence of homopeptides or low-complexity regions (i.e., regions that are made from a small subset of amino-acid residue types). The most prevalent of these are made from residues encoded by adenine/thymidine (AT)-rich codons, in particular asparagine. We examined homopeptide occurrences within protein domains in P. falciparum. Homopeptide enrichments occur for hydrophobic (e.g., valine), or small residues (alanine or glycine) in short spans (

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.

Intrinsically disordered proteins (IDPs) lack stable tertiary structure under physiological conditions. The unique composition and complex dynamical behaviour of IDPs make them a challenge for structural biology and molecular evolution studies. Using NMR ensembles, we found that IDPs evolve under a strong site-specific evolutionary rate heterogeneity, mainly originated by different constraints derived from their inter-residue contacts. Evolutionary rate profiles correlate with the experimentally observed conformational diversity of the protein, allowing the description of different conformational patterns possibly related to their structure-function relationships. The correlation between evolutionary rates and contact information improves when structural information is taken not from any individual conformer or the whole ensemble, but from combining a limited number of conformers. Our results suggest that residue contacts in disordered regions constrain evolutionary rates to conserve the dynamic behaviour of the ensemble and that evolutionary rates can be used as a proxy for the conformational diversity of IDPs.Significance StatementIntrinsically disordered proteins (IDPs) challenge the structure-function relationship paradigm. In this work we found that individual sites of IDPs evolve under a strong rate heterogeneity, mainly due to the structural constraints imposed by contacts between their residues. This can be better explained if the contacts are taken from selected subsets of their alternative native conformations, rather than from individual conformations or the whole native ensemble. From an evolutionary point of view, this result indicates that experimentally-based ensembles are redundant. We also observed that the evolutionary rates follow the structural variability between conformers, unveiling conformational preferences. Our results set the stage for establishing novel evolutionary-based methods to study IDP ensembles.

Viral BCL2 proteins (vBCL2s) help to sustain chronic infection of host proteins to inhibit apoptosis and autophagy. However, details of conformational changes in vBCL2s that enable binding to BH3Ds remain unknown. Using all-atom, multiple microsecond-long molecular dynamic simulations (totaling 17 &mu, s) of the murine &gamma, herpesvirus 68 vBCL2 (M11), and statistical inference techniques, we show that regions of M11 transiently unfold and refold upon binding of the BH3D. Further, we show that this partial unfolding/refolding within M11 is mediated by a network of hydrophobic interactions, which includes residues that are 10 &Aring, away from the BH3D binding cleft. We experimentally validate the role of these hydrophobic interactions by quantifying the impact of mutating these residues on binding to the Beclin1/BECN1 BH3D, demonstrating that these mutations adversely affect both protein stability and binding. To our knowledge, this is the first study detailing the binding-associated conformational changes and presence of long-range interactions within vBCL2s.

Cell communication and signaling 18(1), 1-30 (2020). doi:10.1186/s12964-020-00626-6, Background:Class 1 cytokine receptors (C1CRs) are single-pass transmembrane proteins responsible for transmitting signals between the outside and the inside of cells. Remarkably, they orchestrate key biological processes such as proliferation, differentiation, immunity and growth through long disordered intracellular domains (ICDs), but without having intrinsic kinase activity. Despite these key roles, their characteristics remain rudimentarily understood.Methods:The current paper asks the question of why disorder has evolved to govern signaling of C1CRs by reviewing the literature in combination with new sequence and biophysical analyses of chain properties across the family.Results:We uncover that the C1CR-ICDs are fully disordered and brimming with SLiMs. Many of these short linear motifs (SLiMs) are overlapping, jointly signifying a complex regulation of interactions, including network rewiring by isoforms. The C1CR-ICDs have unique properties that distinguish them from most IDPs and we forward the perception that the C1CR-ICDs are far from simple strings with constitutively bound kinases. Rather, they carry both organizational and operational features left uncovered within their disorder, including mechanisms and complexities of regulatory functions.Conclusions:Critically, the understanding of the fascinating ability of these long, completely disordered chains to orchestrate complex cellular signaling pathways is still in its infancy, and we urge a perceptional shift away from the current simplistic view towards uncovering their full functionalities and potential., Published by Biomed Central, London

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.

Background Proteins often contain regions that are compositionally biased (CB), i.e., they are made from a small subset of amino-acid residue types. These CB regions can be functionally important, e.g., the prion-forming and prion-like regions that are rich in asparagine and glutamine residues. Results Here I report a new program fLPS that can rapidly annotate CB regions. It discovers both single-residue and multiple-residue biases. It works through a process of probability minimization. First, contigs are constructed for each amino-acid type out of sequence windows with a low degree of bias; second, these contigs are searched exhaustively for low-probability subsequences (LPSs); third, such LPSs are iteratively assessed for merger into possible multiple-residue biases. At each of these stages, efficiency measures are taken to avoid or delay probability calculations unless/until they are necessary. On a current desktop workstation, the fLPS algorithm can annotate the biased regions of the yeast proteome (>5700 sequences) in 65 million sequences) in as little as ~1 h, which is >2 times faster than the commonly used program SEG, using default parameters. fLPS discovers both shorter CB regions (of the sort that are often termed ‘low-complexity sequence’), and milder biases that may only be detectable over long tracts of sequence. Conclusions fLPS can readily handle very large protein data sets, such as might come from metagenomics projects. It is useful in searching for proteins with similar CB regions, and for making functional inferences about CB regions for a protein of interest. The fLPS package is available from: http://biology.mcgill.ca/faculty/harrison/flps.html, or https://github.com/pmharrison/flps, or is a supplement to this article. Electronic supplementary material The online version of this article (10.1186/s12859-017-1906-3) contains supplementary material, which is available to authorized users.

Protein turnover is a key aspect of cellular homeostasis and proteome dynamics. However, there is little consensus on which properties of a protein determine its lifetime in the cell. In this work, we exploit two reliable datasets of experimental protein degradation rates to learn models and uncover determinants of protein degradation, with particular focus on properties that can be derived from the sequence. Our work shows that simple sequence features suffice to obtain predictive models of which the output correlates reasonably well with the experimentally measured values. We also show that intrinsic disorder may have a larger effect than previously reported, and that the effect of PEST regions, long thought to act as specific degradation signals, can be better explained by their disorder. We also find that determinants of protein degradation depend on the cell types or experimental conditions studied. This analysis serves as a first step towards the development of more complex, mature computational models of degradation of proteins and eventually of their full life cycle. Proteins 2017; 85:1593-1601. © 2017 Wiley Periodicals, Inc.

Hundreds of billions of dollars have been spent for over three decades in the search for an effective human immunodeficiency virus (HIV) vaccine with no success. There are also at least two other sexually transmitted viruses, for which no vaccine is available, the herpes simplex virus (HSV) and the hepatitis C virus (HCV). Traditional textbook explanatory paradigm of rapid mutation of retroviruses cannot adequately address the unavailability of vaccine for many sexually transmissible viruses, since HSV and HCV are DNA and non-retroviral RNA viruses, respectively, whereas effective vaccine for the horsefly-transmitted retroviral cousin of HIV, equine infectious anemia virus (EIAV), was found in 1973. We reported earlier the highly disordered nature of proteins in outer shells of the HIV, HCV, and HSV. Such levels of disorder are completely absent among the classical viruses, such as smallpox, rabies, yellow fever, and polio viruses, for which efficient vaccines were discovered. This review analyzes the physiology and shell disorder of the various related and non-related viruses to argue that EIAV and the classical viruses need harder shells to survive during harsher conditions of non-sexual transmissions, thus making them vulnerable to antibody detection and neutralization. In contrast, the outer shell of the HIV-1 (with its preferential sexual transmission) is highly disordered, thereby allowing large scale motions of its surface glycoproteins and making it difficult for antibodies to bind to them. The theoretical underpinning of this concept is retrospectively traced to a classical 1920s experiment by the legendary scientist, Oswald Avery. This concept of viral shapeshifting has implications for improved treatment of cancer and infections via immune evasion.

The dual-family peptidylprolyl cis-trans isomerases (immunophilins) represent a naturally occurring chimera of the classical FK506-binding protein (FKBP) and cyclophilin (CYN), connected by a flexible linker. They are found exclusively in monocellular organisms. The modular builds of these molecules represent two distinct types: CYN-(linker)-FKBP and FKBP-3TPR (tetratricopeptide repeat)-CYN. Abbreviated respectively as CFBP and FCBP, the two classes also exhibit distinct organism preference, the CFBP being found in prokaryotes, and the FCBP in eukaryotes. This review summarizes the mystery of these unique class of prolyl isomerases, focusing on their host organisms, potential physiological role, and likely routes of evolution.

Conformational protein properties are coupled to protein functionality and could provide a useful parameter for functional annotation of differentially expressed genes in transcriptome studies. The aim was to determine whether predicted intrinsic protein disorder was differentially associated with proteins encoded by genes that are differentially regulated in lymphoma cells upon interaction with stromal cells, an interaction that occurs in microenvironments, such as lymph nodes that are protective for lymphoma cells during chemotherapy. Intrinsic disorder protein properties were extracted from the Database of Disordered Protein Prediction (D2P2), which contains data from nine intrinsic disorder predictors. Proteins encoded by differentially regulated cell-adhesion regulated genes were enriched in intrinsically disordered regions (IDRs) compared to other genes both with regard to IDR number and length. The enrichment was further ascribed to down-regulated genes. Consistently, a higher proportion of proteins encoded by down-regulated genes contained at least one IDR or were completely disordered. We conclude that down-regulated genes in stromal cell-adherent lymphoma cells encode proteins that are characterized by elevated levels of intrinsically disordered conformation, indicating the importance of down-regulating functional mechanisms associated with intrinsically disordered proteins in these cells. Further, the approach provides a generally applicable and complementary alternative to classification of differentially regulated genes using gene ontology or pathway enrichment analysis.

Calcium is one of the most important second messengers and its intracellular signaling regulates many aspects of cell physiology. Calcium ions, like phosphate ions, are highly charged and thus are able to alter protein conformation upon binding; thereby they constitute key factors in signal transduction. One of the most common calcium-binding structural motifs is the EF-hand, a well-defined helix-loop-helix structural domain, present in many calcium-binding proteins (CBPs). Nonetheless, some CBPs contain non-canonical, disordered motifs, which usually bind calcium with high capacity and low affinity, and which represent a subset of proteins with specific functions, but these functions rarely involve signaling. When compared with phosphorylation-mediated signal transduction, the role of intrinsic disorder in calcium signaling is significantly less prominent and not direct. The list of known examples of intrinsically disordered CBPs is relatively short and the disorder in these examples seems to be linked to secretion and storage. Calcium-sensitive phosphatase calcineurin is an exception, but it represents an example of transient disorder, which is, nevertheless, vital to the functioning of this protein. The underlying reason for the different role of disordered proteins in the two main cellular signaling systems appears to be linked to the gradient of calcium concentration, present in all living cells.

The inhibitor of DNA binding and cell differentiation 2 (Id2) is a helix-loop-helix (HLH) protein that acts as negative dominant regulator of basic-HLH transcription factors during development and in cancer. The structural properties of Id2 have been investigated so far by using synthetic or recombinant fragments reproducing single domains (N-terminus, HLH, C-terminus): the HLH domain tends to dimerize into a four-helix bundle, whereas the flanking regions are flexible. In this work, the intact protein was expressed in E. coli, solubilized from inclusion bodies with urea, purified and dissolved in water at pH~4. Under these conditions, Id2 was obtained with both cysteine residues disulfide-bonded to β-mercaptoethanol that was present during the solubilization process. Moreover, it existed in a self-assembled state, in which the N-terminus remained highly flexible, while the HLH domain and, surprisingly, part of the C-terminus, which corresponds to the nuclear export signal (NES), both were involved in slowly tumbling, rigid structures. The protein oligomers also formed twisted fibrils that were several micrometers long and up to 80 nm thick. These results show that self-assembly decreases the backbone flexibility of those two protein regions (HLH and NES) that are important for interaction with basic-HLH transcription factors or for nucleocytoplasmic shuttling.

We provide first large scale analysis of the peculiarities of surface areas of 5658 dissimilar (below 50% sequence similarity) proteins with known 3D-structures that bind to proteins, DNA or RNAs. We show here that area of the protein surface is highly correlated with the protein length. The size of the interface surface is only modestly correlated with the protein size, except for RNA-binding proteins where larger proteins are characterized by larger interfaces. Disordered proteins with disordered interfaces are characterized by significantly larger per-residue areas of their surfaces and interfaces when compared to the structured proteins. These result are applicable for proteins involved in interaction with DNA, RNA, and proteins and suggest that disordered proteins and binding regions are less compact and more likely to assume extended shape. We demonstrate that disordered protein binding residues in the interfaces of disordered proteins drive the increase in the per residue area of these interfaces. Our results can be used to predict in silico whether a given protomer from the DNA, RNA or protein complex is likely to be disordered in its unbound form.