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In tumors, mutation in Ras proteins stimulates a signaling cascade through phosphorylation. Downstream of the cascade, many transcription and translation factors are up- or down-regulated by phosphorylation, leading to cancer progression. This phosphorylation cascade is sustained by 14-3-3ζ protein. 14-3-3ζ binds to its client proteins that are Ser/Thr-phosphorylated and prevents their dephosphorylation. One of those transcription factors is FOXO3a, whose transcriptional activity is suppressed in the phosphorylation cascade. FOXO3a binds to specific DNA sequences and activates the transcription of apoptosis-related proteins. In cancer cells, however, FOXO3a is phosphorylated, bound to 14-3-3ζ, and dissociated from the DNA, resulting in FOXO3a inactivation. To elucidate the mechanism of FOXO3a inactivation by the 14-3-3ζ binding, we aim to perform NMR analysis of the interaction between 14-3-3ζ and di-phosphorylated FOXO3a residues 1-284 (dpFOXO3a). Here, we report the backbone resonance assignments of dpFOXO3a, which are transferred from those of the N-terminal domain (NTD) and the DNA-binding domain (DBD) of dpFOXO3a.

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The diagnostic odysseys for rare disease patients are getting shorter as next-generation sequencing becomes more widespread. However, the complex genetic diversity and factors influencing expressivity continue to challenge accurate diagnosis, leaving more than 50% of genetic variants categorized as variants of uncertain significance.Genomic expression intricately hinges on localized interactions among its products. Conventional variant prioritization, biased towards known disease genes and the structure-function paradigm, overlooks the potential impact of variants shaping the composition, location, size, and properties of biomolecular condensates, genuine membraneless organelles swiftly sensing and responding to environmental changes, and modulating expressivity.To address this complexity, we propose to focus on the nexus of genetic variants within biomolecular condensates determinants. Scrutinizing variant effects in these membraneless organelles could refine prioritization, enhance diagnostics, and unveil the molecular underpinnings of rare diseases. Integrating comprehensive genome sequencing, transcriptomics, and computational models can unravel variant pathogenicity and disease mechanisms, enabling precision medicine. This paper presents the rationale driving our proposal and describes a protocol to implement this approach. By fusing state-of-the-art knowledge and methodologies into the clinical practice, we aim to redefine rare diseases diagnosis, leveraging the power of scientific advancement for more informed medical decisions.

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Arginyltransferase 1 (ATE1) catalyzes arginylation, an important post-translational modification (PTM) in eukaryotes that plays a critical role in cellular homeostasis. The disruption of ATE1 function is implicated in mammalian neurodegenerative disorders and cardiovascular maldevelopment, while post-translational arginylation has also been linked to the activities of several important human viruses such as SARS-CoV-2 and HIV. Despite the known significance of ATE1 in mammalian cellular function, past biophysical studies of this enzyme have mainly focused on yeast ATE1, leaving the mechanism of arginylation in mammalian cells unclear. In this study, we sought to structurally and biophysically characterize mouse (Mus musculus) ATE1. Using size-exclusion chromatography (SEC), small angle X-ray scattering (SAXS), and hydrogen deuterium exchange mass spectrometry (HDX-MS), assisted by AlphaFold modeling, we found that mouse ATE1 is structurally more complex than yeast ATE1. Importantly, our data indicate the existence of an intrinsically disordered region (IDR) in all mouse ATE1 splice variants. However, comparative HDX-MS analyses show that yeast ATE1 does not have such an IDR, consistent with prior X-ray, cryo-EM, and SAXS analyses. Furthermore, bioinformatics approaches reveal that mammalian ATE1 sequences, as well as in a large majority of other eukaryotes, contain an IDR-like sequence positioned in proximity to the ATE1 GNAT active-site fold. Computational analysis suggests that the IDR likely facilitates the formation of the complex between ATE1 and tRNAArg, adding a new complexity to ATE1 structure and providing new insights for future studies of ATE1 functions.

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Proteins with intrinsically disordered regions (IDRs) often undergo phase separation to control their functions spatiotemporally. Changing the pH alters the protonation levels of charged sidechains, which in turn affects the attractive or repulsive force for phase separation. In a cell, the rupture of membrane-bound compartments, such as lysosomes, creates an abrupt change in pH. However, how proteins' phase separation reacts to different pH environments remains largely unexplored. Here, using extensive mutagenesis, NMR spectroscopy, and biophysical techniques, it is shown that the assembly of galectin-3, a widely studied lysosomal damage marker, is driven by cation-π interactions between positively charged residues in its folded domain with aromatic residues in the IDR in addition to π-π interaction between IDRs. It is also found that the sole two negatively charged residues in its IDR sense pH changes for tuning the condensation tendency. Also, these two residues may prevent this prion-like IDR domain from forming rapid and extensive aggregates. These results demonstrate how cation-π, π-π, and electrostatic interactions can regulate protein condensation between disordered and structured domains and highlight the importance of sparse negatively charged residues in prion-like IDRs.

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The zinc-finger antiviral protein (ZAP) is an innate immunity sensor of non-self nucleic acids. Its antiviral activity is exerted through the physical interaction with different cofactors, including TRIM25, Riplet and KHNYN. Cellular proteins that interact with infectious agents are expected to be engaged in genetic conflicts that often result in their rapid evolution. To test this possibility and to identify the regions most strongly targeted by natural selection, we applied in silico molecular evolution tools to analyze the evolutionary history of ZAP and cofactors in four mammalian groups. We report evidence of positive selection in all genes and in most mammalian groups. On average, the intrinsically disordered regions (IDRs) embedded in the four proteins evolve significantly faster than folded domains and most positively selected sites fall within IDRs. In ZAP, the PARP domain also shows abundant signals of selection, and independent evolution in different mammalian groups suggests modulation of its ADP-ribose binding ability. Detailed analyses of the biophysical properties of IDRs revealed that chain compaction and conformational entropy are conserved across mammals. The IDRs in ZAP and KHNYN are particularly compact, indicating that they may promote phase separation (PS). In line with this hypothesis, we predicted several PS-promoting regions in ZAP and KHNYN, as well as in TRIM25. Positively selected sites are abundant in these regions, suggesting that PS may be important for the antiviral functions of these proteins and the evolutionary arms race with viruses. Our data shed light into the evolution of ZAP and cofactors and indicate that IDRs represent central elements in host-pathogen interactions.

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Intrinsically disordered proteins (IDPs) are emerging therapeutic targets for human diseases. However, probing their transient conformations remains challenging because of conformational heterogeneity. To address this problem, we developed a biosensor using a point-functionalized silicon nanowire (SiNW) that allows for real-time sampling of single-molecule dynamics. A single IDP, N-terminal transactivation domain of tumor suppressor protein p53 (p53TAD1), was covalently conjugated to the SiNW through chemical engineering, and its conformational transition dynamics was characterized as current fluctuations. Furthermore, when a globular protein ligand in solution bound to the targeted p53TAD1, protein-protein interactions could be unambiguously distinguished from large-amplitude current signals. These proof-of-concept experiments enable semiquantitative, realistic characterization of the structural properties of IDPs and constitute the basis for developing a valuable tool for protein profiling and drug discovery in the future.

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The aggregation of the protein α-synuclein into amyloid deposits is associated with multiple neurological disorders, including Parkinson's disease. Soluble amyloid oligomers are reported to exhibit higher toxicity than insoluble amyloid fibrils, with dimers being the smallest toxic oligomer. Small molecule drugs, such as fasudil, have shown potential in targeting α-synuclein aggregation and reducing its toxicity. In this study, we use atomistic molecular dynamics simulations to demonstrate how fasudil affects the earliest stage of aggregation, namely dimerization. Our results show that the presence of fasudil reduces the propensity for intermolecular contact formation between protein chains. Consistent with previous reports, our analysis confirms that fasudil predominantly interacts with the negatively charged C-terminal region of α-synuclein. However, we also observe transient interactions with residues in the charged N-terminal and hydrophobic NAC regions. Our simulations indicate that while fasudil prominently interacts with the C-terminal region, it is the transient interactions with residues in the N-terminal and NAC regions that effectively block the formation of intermolecular contacts between protein chains and prevent early dimerization of this disordered protein.

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Motivation: Intrinsically disordered regions (IDRs) of proteins exist as an ensemble of conformations, and not as a single structure. Existing databases contain extensive, experimentally derived annotations of intrinsic disorder for millions of proteins at the sequence level. However, only a tiny fraction of these IDRs are associated with an experimentally determined protein structure. Moreover, even if a structure exists, parts of the disordered regions may still be unresolved. Results: Here we organize Structures of Intrinsically Disordered Regions (StrIDR), a database of IDRs confirmed via experimental or homology-based evidence, resolved in experimentally determined structures. The database can provide useful insights into the dynamics, folding, and interactions of IDRs. It can also facilitate computational studies on IDRs, such as those using molecular dynamics simulations and/or machine learning. Availability: StrIDR is available at https://isblab.ncbs.res.in/stridr. The web UI allows for downloading PDB structures and SIFTS mappings of individual entries. Additionally, the entire database can be downloaded in a JSON format. The source code for creating and updating the database is available at https://github.com/isblab/stridr.

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Prolyl-hydroxylation is an oxygen-dependent posttranslational modification (PTM) that is known to regulate fibril formation of collagenous proteins and modulate cellular expression of hypoxia-inducible factor (HIF) α subunits. However, our understanding of this important but relatively rare PTM has remained incomplete due to the lack of biophysical methodologies that can directly measure multiple prolyl-hydroxylation events within intrinsically disordered proteins. Here, we describe a real-time 13C-direct detection NMR-based assay for studying the hydroxylation of two evolutionarily conserved prolines (P402 and P564) simultaneously in the intrinsically disordered oxygen-dependent degradation domain of hypoxic-inducible factor 1α by exploiting the "proton-less" nature of prolines. We show unambiguously that P564 is rapidly hydroxylated in a time-resolved manner while P402 hydroxylation lags significantly behind that of P564. The differential hydroxylation rate was negligibly influenced by the binding affinity to prolyl-hydroxylase enzyme, but rather by the surrounding amino acid composition, particularly the conserved tyrosine residue at the +1 position to P564. These findings support the unanticipated notion that the evolutionarily conserved P402 seemingly has a minimal impact in normal oxygen-sensing pathway.

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The class of intrinsically disordered proteins lacks stable three-dimensional structures. Their flexibility allows them to engage in a wide variety of interactions with other biomolecules thus making them biologically relevant and efficient. The intrinsic disorders of these proteins, which undergo binding-induced folding, allow alterations in their topologies while conserving their binding sites. Due to the lack of well-defined three-dimensional structures in the absence of their physiological partners, the folding and the conformational dynamics of these proteins remained poorly understood. Particularly, it is unclear how these proteins exist in the crowded intracellular milieu. In the present study, molecular dynamic simulations of two intrinsically unstructured proteins and two controls (folded proteins) were conducted in the presence and absence of molecular crowders to obtain an in-depth insight into their conformational flexibility. The present study revealed that polymer crowders stabilize the disordered proteins through enthalpic as well as entropic effects that are significantly more than their monomeric counterpart. Taken together, the study delves deep into crowding effects on intrinsically disordered proteins and provides insights into how molecular crowders induce a significantly diverse ensemble of dynamic scaffolds needed to carry out diverse functions.Communicated by Ramaswamy H. Sarma.

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The nuclear pore complex (NPC) is a critical gateway regulating molecular transport between the nucleus and cytoplasm. It allows small molecules to pass freely, while larger molecules require nuclear transport receptors to traverse the barrier. This selective permeability is maintained by phenylalanine-glycine-rich nucleoporins (FG-Nups), intrinsically disordered proteins that fill the NPC's central channel. The disordered and flexible nature of FG-Nups complicates their spatial characterization with conventional structural biology techniques. To address this challenge, polymer physics offers a valuable framework for describing FG-Nup behavior, reducing their complex structures to a few key parameters. In this review, we explore how polymer physics models FG-Nups using these parameters and discuss experimental efforts to quantify them in various contexts, providing insights into the conformational properties of FG-Nups.

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Treacher Collins syndrome (TCS) is a genetic disorder affecting facial development, primarily caused by mutations in the TCOF1 gene. TCOF1, along with NOLC1, play important roles in ribosomal RNA transcription and processing. Previously, a zebrafish model of TCS successfully recapitulated the main characteristics of the syndrome by knocking down the expression of a gene on chromosome 13 (coding for Uniprot ID B8JIY2), which was identified as the TCOF1 orthologue. However, database updates renamed this gene as nolc1 and the zebrafish database (ZFIN) identified a different gene on chromosome 14 as the TCOF1 orthologue (coding for Uniprot ID E7F9D9). NOLC1 and TCOF1 are large proteins with unstructured regions and repetitive sequences that complicate alignments and comparisons. Also, the additional whole genome duplication of teleosts sets further difficulty. In this study, we present evidence that endorses that NOLC1 and TCOF1 are paralogs, and that the zebrafish gene on chromosome 14 is a low-complexity LisH domain-containing factor that displays homology to NOLC1 but lacks essential sequence features to accomplish TCOF1 nucleolar functions. Our analysis also supports the idea that zebrafish, as has been suggested for other non-tetrapod vertebrates, lack the TCOF1 gene that is associated with tripartite nucleolus. Using BLAST searches in a group of teleost genomes, we identified fish-specific sequences similar to E7F9D9 zebrafish protein. We propose naming them "LisH-containing Low Complexity Proteins" (LLCP). Interestingly, the gene on chromosome 13 (nolc1) displays the sequence features, developmental expression patterns, and phenotypic impact of depletion that are characteristic of TCOF1 functions. These findings suggest that in teleost fish, the nucleolar functions described for both NOLC1 and TCOF1 mediated by their repeated motifs, are carried out by a single gene, nolc1. Our study, which is mainly based on computational tools available as free web-based algorithms, could help to solve similar conflicts regarding gene orthology in zebrafish.

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Selective compartmentalization of cellular contents is fundamental to the regulation of biochemistry. Although membrane-bound organelles control composition by using a semi-permeable barrier, biomolecular condensates rely on interactions among constituents to determine composition. Condensates are formed by dynamic multivalent interactions, often involving intrinsically disordered regions (IDRs) of proteins, yet whether distinct compositions can arise from these dynamic interactions is not known. Here, by comparative analysis of proteins differentially partitioned by two different condensates, we find that distinct compositions arise through specific IDR-mediated interactions. The IDRs of differentially partitioned proteins are necessary and sufficient for selective partitioning. Distinct sequence features are required for IDRs to partition, and swapping these sequence features changes the specificity of partitioning. Swapping whole IDRs retargets proteins and their biochemical activity to different condensates. Our results demonstrate that IDR-mediated interactions can target proteins to specific condensates, enabling the spatial regulation of biochemistry within the cell.

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Ribosomal protein SA (RPSA) plays multiple roles in cells, including ribosomal biogenesis and translation, cellular migration, and cytoskeleton reorganization. RPSA is crucial in the process of pathogen infection. Extensive research has examined RPSA's role in pathogen adhesion and invasion, but its broader functions, particularly its anti-infective capabilities, have garnered increasing attention in recent years. This dual role is closely related to its structural domains, which influence its localization and function. This review summarizes key research findings concerning the functional domains of RPSA and analyzes the relationship between its membrane localization and structural domains. Additionally, the functional implications of RPSA are categorized based on its different localizations during pathogen infection. Specifically, when RPSA is located on the cell surface, it promotes pathogen adhesion and invasion of host cells; conversely, when RPSA is located intracellularly, it exhibits anti-infective properties. Overall, RPSA shows a dual nature, both in facilitating pathogen invasion of the host and in possessing the ability to resist pathogen infection. This review comprehensively examines the dual role of RPSA in pathogen infection by analyzing its structural domains, localization, and interactions with cellular and pathogen molecules. Our aim is to update and deepen researchers' understanding of the various functions of RPSA during pathogen infection.

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Linker histones play an essential role in chromatin packaging by facilitating compaction of the 11-nm fiber of nucleosomal "beads on a string." The result is a heterogeneous condensed state with local properties that range from dynamic, irregular, and liquid-like to stable and regular structures (the 30-nm fiber), which in turn impact chromatin-dependent activities at a fundamental level. The properties of the condensed state depend on the type of linker histone, particularly on the highly disordered C-terminal tail, which is the most variable region of the protein, both between species, and within the various subtypes and cell-type specific variants of a given organism. We have developed an in vitro model system comprising linker histone tail and linker DNA, which although very minimal, displays surprisingly complex behavior, and is sufficient to model the known states of linker histone-condensed chromatin: disordered "fuzzy" complexes ("open" chromatin), dense liquid-like assemblies (dynamic condensates), and higher-order structures (organized 30-nm fibers). A crucial advantage of such a simple model is that it allows the study of the various condensed states by NMR, circular dichroism, and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. We have leveraged this to rationalize the distinct condensing properties of linker histone subtypes and variants across species that are encoded by the amino acid content of their C-terminal tails. Three properties emerge as key to defining the condensed state: charge density, lysine/arginine ratio, and proline-free regions, and we evaluate each separately using a strategic mutagenesis approach.

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Brain functioning relies on orchestrated synaptic vesicle dynamics and controlled neurotransmitter release. Multiple biomolecular condensates coexist at the pre- and post-synapse and they are driven by condensation that combines binding, phase separation, and percolation. In pre-synapses, intrinsically disordered regions (IDRs) of synaptic proteins are drivers of condensation that enable clustering of synaptic vesicles (SVs). Although sequences of IDRs are poorly conserved across evolution, our computational analysis reveals the existence of non-random compositional biases and sequence patterns (molecular grammars) in IDRs of pre-synaptic proteins. For example, synapsin-1, which is essential for condensation of SVs, contains a conserved valence of arginine residues and blocks of polar and proline residues that are segregated from one another along the linear sequence. We show that these conserved features are crucial for driving synapsin-1 condensation in vitro and in cells. Our results highlight how conserved molecular grammars drive the condensation of key proteins at the pre-synapse.

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Plant hormone-related transcription factors (TFs) are key regulators of plant development, responses to environmental stress such as climate changes, pathogens, and pests. These TFs often function as families that exhibit genetic redundancy in higher plants, and are affected by complex crosstalk mechanisms between different plant hormones. These properties make it difficult to analyze and control them in many cases. In this study, we introduced a chemical inhibitor to manipulate plant hormone-related TFs, focusing on the jasmonate (JA) and ethylene (ET) signaling pathways, with the key TFs MYC2/3/4 and EIN3/EIL1. This study revealed that JAZ10CMID, the binding domain of the repressor involved in the desensitization of both TFs, is an intrinsically disordered region in the absence of binding partners. Chemical inhibitors have been designed based on this interaction to selectively inhibit MYC TFs while leaving EIN3/EIL1 unaffected. This peptide inhibitor effectively disrupts MYC-mediated responses while activating EIN3-mediated responses and successfully uncouples the crosstalk between JA and ET signaling in Arabidopsis thaliana. Furthermore, the designed peptide inhibitor was also shown to selectively inhibit the activity of MpMYC, an ortholog of AtMYC in Marchantia polymorpha, demonstrating its applicability across different plant species. This underscores the potential of using peptide inhibitors for specific TFs to elucidate hormone crosstalk mechanisms in non-model plants without genetic manipulation. Such a design concept for chemical fixation of the disordered structure is expected to limit the original multiple binding partners and provide useful chemical tools in chemical biology research.

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Several mammalian genes have originated from the domestication of retrotransposons, selfish mobile elements related to retroviruses. Some of the proteins encoded by these genes have maintained virus-like features; including self-processing, capsid structure formation, and the generation of different isoforms through -1 programmed ribosomal frameshifting. Using quantitative approaches in molecular evolution and biophysical analyses, we studied 28 retrotransposon-derived genes, with a focus on the evolution of virus-like features. By analyzing the rate of synonymous substitutions, we show that the -1 programmed ribosomal frameshifting mechanism in three of these genes (PEG10, PNMA3, and PNMA5) is conserved across mammals and originates alternative proteins. These genes were targets of positive selection in primates, and one of the positively selected sites affects a B-cell epitope on the spike domain of the PNMA5 capsid, a finding reminiscent of observations in infectious viruses. More generally, we found that retrotransposon-derived proteins vary in their intrinsically disordered region content and this is directly associated with their evolutionary rates. Most positively selected sites in these proteins are located in intrinsically disordered regions and some of them impact protein posttranslational modifications, such as autocleavage and phosphorylation. Detailed analyses of the biophysical properties of intrinsically disordered regions showed that positive selection preferentially targeted regions with lower conformational entropy. Furthermore, positive selection introduces variation in binary sequence patterns across orthologues, as well as in chain compaction. Our results shed light on the evolutionary trajectories of a unique class of mammalian genes and suggest a novel approach to study how intrinsically disordered region biophysical characteristics are affected by evolution.

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RTEL1 is an essential DNA helicase which plays an important role in various aspects of genome stability, from telomere metabolism to DNA replication, repair and recombination. RTEL1 has been implicated in a number of genetic diseases and cancer development, including glioma, breast, lung and gastrointestinal tumors. RTEL1 is a FeS helicase but, in addition to the helicase core, it comprises a long C-terminal region which includes a number of folded domains connected by intrinsically disordered loops and mediates RTEL1 interaction with factors involved in pivotal cellular pathways. However, information on the architecture and the function of this region is still limited. We expressed and purified a variety of fragments encompassing the folded domains and the unstructured regions. We determined the crystal structure of the second repeat, confirming that it has a fold similar to the harmonin homology domains. SAXS data provide low-resolution information on all the fragments and suggest that the presence of the RING domain affects the overall architecture of the C-terminal region, making the structure significantly more compact. NMR data provide experimental information on the interaction between PCNA and the RTEL1 C-terminal region, revealing a putative low-affinity additional site of interaction. A biochemical analysis shows that the C-terminal region, in addition to a preference for telomeric RNA and DNA G-quadruplexes, has a high affinity for R-loops and D-loops, consistent with the role played by the RTEL1 helicase in homologous recombination, telomere maintenance and preventing replication-transcription conflicts. We further dissected the contribution of each domain in binding different substrates.

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The Nipah and Hendra viruses are severe human pathogens. In addition to the P protein, their P gene also encodes the V and W proteins that share with P their N-terminal intrinsically disordered domain (NTD) and possess distinct C-terminal domains (CTDs). The W protein is a key player in the evasion of the host innate immune response. We previously showed that the W proteins are intrinsically disordered and can form amyloid-like fibrils. However, structural information on W CTD (CTDW) and its potential contribution to the fibrillation process is lacking. In this study, we demonstrate that CTDWS are disordered and able to form dimers mediated by disulfide bridges. We also show that the NTD and the CTDW interact with each other and that this interaction triggers both a gain of secondary structure and a chain compaction within the NTD. Finally, despite the lack of intrinsic fibrillogenic properties, we show that the CTDW favors the formation of fibrils by the NTD both in cis and in trans. Altogether, the results herein presented shed light on the molecular mechanisms underlying Henipavirus pathogenesis and may thus contribute to the development of targeted therapies.