PAICS ubiquitination recruits UBAP2 to trigger phase separation for purinosome assembly.

Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies.

Musashi-1: An Example of How Polyalanine Tracts Contribute to Self-Association in the Intrinsically Disordered Regions of RNA-Binding Proteins.

RNA-binding proteins (RBPs) have intrinsically disordered regions (IDRs) whose biophysical properties have yet to be explored to the same extent as those of the folded RNA interacting domains. These IDRs are essential to the formation of biomolecular condensates, such as stress and RNA granules, but dysregulated assembly can be pathological. Because of their structural heterogeneity, IDRs are best studied by NMR spectroscopy. In this study, we used NMR spectroscopy to investigate the structural propensity and self-association of the IDR of the RBP Musashi-1. We identified two transient α-helical regions (residues ~208-218 and ~270-284 in the IDR, the latter with a polyalanine tract). Strong NMR line broadening in these regions and circular dichroism and micrography data suggest that the two α-helical elements and the hydrophobic residues in between may contribute to the formation of oligomers found in stress granules and implicated in Alzheimer's disease. Bioinformatics analysis suggests that polyalanine stretches in the IDRs of RBPs may have evolved to promote RBP assembly.

Interactions between the Intrinsically Disordered Regions of hnRNP-A2 and TDP-43 Accelerate TDP-43's Conformational Transition.

Most biological functions involve protein-protein interactions. Our understanding of these interactions is based mainly on those of structured proteins, because encounters between intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are much less studied, regardless of the fact that more than half eukaryotic proteins contain IDRs. RNA-binding proteins (RBPs) are a large family whose members almost all have IDRs in addition to RNA binding domains. These IDRs, having low sequence similarity, interact, but structural details on these interactions are still lacking. Here, using the IDRs of two RBPs (hnRNA-A2 and TDP-43) as a model, we demonstrate that the rate at which TDP-43's IDR undergoes the neurodegenerative disease related α-helix-to-ß-sheet transition increases in relation to the amount of hnRNP-A2's IDR that is present. There are more than 1500 RBPs in human cells and most of them have IDRs. RBPs often join the same complexes to regulate genes. In addition to the structured RNA-recognition motifs, our study demonstrates a general mechanism through which RBPs may regulate each other's functions through their IDRs.

TAR DNA-binding protein 43 (TDP-43) liquid-liquid phase separation is mediated by just a few aromatic residues.

Eukaryotic cells contain distinct organelles, but not all of these compartments are enclosed by membranes. Some intrinsically disordered proteins mediate membraneless organelle formation through liquid-liquid phase separation (LLPS). LLPS facilitates many biological functions such as regulating RNA stability and ribonucleoprotein assembly, and disruption of LLPS pathways has been implicated in several diseases. Proteins exhibiting LLPS typically have low sequence complexity and specific repeat motifs. These motifs promote multivalent connections with other molecules and the formation of higher-order oligomers, and their removal usually prevents LLPS. The intrinsically disordered C-terminal domain of TAR DNA-binding protein 43 (TDP-43), a protein involved in motor neuron disease and dementia lacks a dominant LLPS motif, however, and how this domain forms condensates is unclear. Using extensive mutagenesis of TDP-43, we demonstrate here that three tryptophan residues and, to a lesser extent, four other aromatic residues are most important for TDP-43 to undergo LLPS. Our results also suggested that only a few residues may be required for TDP-43 LLPS because the α-helical segment (spanning ∼20 residues) in the middle part of the C-terminal domain tends to self-assemble, reducing the number of motifs required for forming a multivalent connection. Our results indicating that a self-associating α-helical element with a few key residues regulates condensate formation highlight a different type of LLPS involving intrinsically disordered regions. The C-terminal domain of TDP-43 contains ∼50 disease-related mutations, with no clear physicochemical link between them. We propose that they may disrupt LLPS indirectly by interfering with the key residues identified here.

Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS.

The properties of unfolded proteins are essential both for the mechanisms of protein folding and for the function of the large group of intrinsically disordered proteins. However, the detailed structural and dynamical characterization of these highly dynamic and conformationally heterogeneous ensembles has remained challenging. Here we combine and compare three of the leading techniques for the investigation of unfolded proteins, NMR spectroscopy (NMR), small-angle X-ray scattering (SAXS), and single-molecule Förster resonance energy transfer (FRET), with the goal of quantitatively testing their consistency and complementarity and for obtaining a comprehensive view of the unfolded-state ensemble. Using unfolded ubiquitin as a test case, we find that its average dimensions derived from FRET and from structural ensembles calculated using the program X-PLOR-NIH based on NMR and SAXS restraints agree remarkably well; even the shapes of the underlying intramolecular distance distributions are in good agreement, attesting to the reliability of the approaches. The NMR-based results provide a highly sensitive way of quantifying residual structure in the unfolded state. FRET-based nanosecond fluorescence correlation spectroscopy allows long-range distances and chain dynamics to be probed in a time range inaccessible by NMR. The combined techniques thus provide a way of optimally using the complementarity of the available methods for a quantitative structural and dynamical description of unfolded proteins both at the global and the local level.

The intrinsically disordered N-terminal domain of galectin-3 dynamically mediates multisite self-association of the protein through fuzzy interactions.

Galectins are a family of lectins that bind ß-galactosides through their conserved carbohydrate recognition domain (CRD) and can induce aggregation with glycoproteins or glycolipids on the cell surface and thereby regulate cell activation, migration, adhesion, and signaling. Galectin-3 has an intrinsically disordered N-terminal domain and a canonical CRD. Unlike the other 14 known galectins in mammalian cells, which have dimeric or tandem-repeated CRDs enabling multivalency for various functions, galectin-3 is monomeric, and its functional multivalency therefore is somewhat of a mystery. Here, we used NMR spectroscopy, mutagenesis, small-angle X-ray scattering, and computational modeling to study the self-association-related multivalency of galectin-3 at the residue-specific level. We show that the disordered N-terminal domain (residues ∼20-100) interacts with itself and with a part of the CRD not involved in carbohydrate recognition (ß-strands 7-9; residues ∼200-220), forming a fuzzy complex via inter- and intramolecular interactions, mainly through hydrophobicity. These fuzzy interactions are characteristic of intrinsically disordered proteins to achieve liquid-liquid phase separation, and we demonstrated that galectin-3 can also undergo liquid-liquid phase separation. We propose that galectin-3 may achieve multivalency through this multisite self-association mechanism facilitated by fuzzy interactions.

The physical forces mediating self-association and phase-separation in the C-terminal domain of TDP-43.

The TAR DNA-binding protein of 43kDa (TDP-43) has been identified as the main component of amyotrophic lateral sclerosis (ALS) cytoplasmic inclusions. The link between this proteinopathy and TDP-43's intrinsically disordered C-terminal domain is well known, but recently also, this domain has been shown to be involved in the formation of the membraneless organelles that mediate TDP-43's functions. The mechanisms that underpin the liquid-liquid phase separation (LLPS) of these membraneless organelles undergo remain elusive. Crucially though, these factors may be the key to understanding the delicate balance between TDP-43's physiological and pathological functions. In this study, we used nuclear magnetic resonance spectroscopy and optical methods to demonstrate that an α-helical component in the centre (residues 320-340) of the C-terminal domain is related to the protein's self-association and LLPS. Systematically analysing ALS-related TDP-43 mutants (G298S, M337V, and Q331K) in different buffer conditions at different temperatures, we prove that this phase separation is driven by hydrophobic interactions but is inhibited by electrostatic repulsion. Based on these findings, we rationally introduced a mutant, W334G, and demonstrate that this mutant disrupts LLPS without disturbing this α-helical propensity. This tryptophan may serve as a key residue in this protein's LLPS.

Transient electrostatic interactions dominate the conformational equilibrium sampled by multidomain splicing factor U2AF65: a combined NMR and SAXS study.

Multidomain proteins containing intrinsically disordered linkers exhibit large-scale dynamic modes that play key roles in a multitude of molecular recognition and signaling processes. Here, we determine the conformational space sampled by the multidomain splicing factor U2AF65 using complementary nuclear magnetic resonance spectroscopy and small-angle scattering data. Available degrees of conformational freedom are initially stochastically sampled and experimental data then used to delineate the potential energy landscape in terms of statistical probability. The spatial distribution of U2AF65 conformations is found to be highly anisotropic, comprising significantly populated interdomain contacts that appear to be electrostatic in origin. This hypothesis is supported by the reduction of signature PREs reporting on expected interfaces with increasing salt concentration. The described spatial distribution reveals the complete spectrum of the unbound forms of U2AF65 that coexist with the small percentage of a preformed RNA-bound domain arrangement required for polypyrimidine-tract recognition by conformational selection. More generally, the proposed approach to describing conformational equilibria of multidomain proteins can be further combined with other experimental data that are sensitive to domain dynamics.

A Few Charged Residues in Galectin-3's Folded and Disordered Regions Regulate Phase Separation.

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.

Galectin-3 aggravates microglial activation and tau transmission in tauopathy.

Alzheimer's disease is characterized by the accumulation of amyloid-ß plaques, aggregation of hyperphosphorylated tau (pTau), and microglia activation. Galectin-3 (Gal3) is a ß-galactoside-binding protein that has been implicated in amyloid pathology. Its role in tauopathy remains enigmatic. Here, we showed that Gal3 was upregulated in the microglia of humans and mice with tauopathy. pTau triggered the release of Gal3 from human induced pluripotent stem cell-derived microglia in both its free and extracellular vesicular-associated (EV-associated) forms. Both forms of Gal3 increased the accumulation of pathogenic tau in recipient cells. Binding of Gal3 to pTau greatly enhanced tau fibrillation. Besides Gal3, pTau was sorted into EVs for transmission. Moreover, pTau markedly enhanced the number of EVs released by iMGL in a Gal3-dependent manner, suggesting a role of Gal3 in biogenesis of EVs. Single-cell RNA-Seq analysis of the hippocampus of a mouse model of tauopathy (THY-Tau22) revealed a group of pathogenic tau-evoked, Gal3-associated microglia with altered cellular machineries implicated in neurodegeneration, including enhanced immune and inflammatory responses. Genetic removal of Gal3 in THY-Tau22 mice suppressed microglia activation, reduced the level of pTau and synaptic loss in neurons, and rescued memory impairment. Collectively, Gal3 is a potential therapeutic target for tauopathy.

Flexible-meccano: a tool for the generation of explicit ensemble descriptions of intrinsically disordered proteins and their associated experimental observables.

MOTIVATION: Intrinsically disordered proteins (IDPs) represent a significant fraction of the human proteome. The classical structure function paradigm that has successfully underpinned our understanding of molecular biology breaks down when considering proteins that have no stable tertiary structure in their functional form. One convenient approach is to describe the protein in terms of an equilibrium of rapidly inter-converting conformers. Currently, tools to generate such ensemble descriptions are extremely rare, and poorly adapted to the prediction of experimental data. RESULTS: We present flexible-meccano-a highly efficient algorithm that generates ensembles of molecules, on the basis of amino acid-specific conformational potentials and volume exclusion. Conformational sampling depends uniquely on the primary sequence, with the possibility of introducing additional local or long-range conformational propensities at an amino acid-specific resolution. The algorithm can also be used to calculate expected values of experimental parameters measured at atomic or molecular resolution, such as nuclear magnetic resonance (NMR) and small angle scattering, respectively. We envisage that flexible-meccano will be useful for researchers who wish to compare experimental data with those expected from a fully disordered protein, researchers who see experimental evidence of deviation from 'random coil' behaviour in their protein, or researchers who are interested in working with a broad ensemble of conformers representing the flexibility of the IDP of interest. AVAILABILITY: A fully documented multi-platform executable is provided, with examples, at http://www.ibs.fr/science-213/scientific-output/software/flexible-meccano/ CONTACT: martin.blackledge@ibs.fr.

Direct prediction of NMR residual dipolar couplings from the primary sequence of unfolded proteins.

Conformational analysis: an approach to the prediction of RDCs from disordered protein chains, integrating the effect of nearest neighbors and the alignment characteristics of the statistical coil, is reported. NMR residual dipolar couplings (RDC) are sensitive probes of conformational sampling in unfolded proteins.

IFF: Identifying key residues in intrinsically disordered regions of proteins using machine learning.

Conserved residues in protein homolog sequence alignments are structurally or functionally important. For intrinsically disordered proteins or proteins with intrinsically disordered regions (IDRs), however, alignment often fails because they lack a steric structure to constrain evolution. Although sequences vary, the physicochemical features of IDRs may be preserved in maintaining function. Therefore, a method to retrieve common IDR features may help identify functionally important residues. We applied unsupervised contrastive learning to train a model with self-attention neuronal networks on human IDR orthologs. Parameters in the model were trained to match sequences in ortholog pairs but not in other IDRs. The trained model successfully identifies previously reported critical residues from experimental studies, especially those with an overall pattern (e.g., multiple aromatic residues or charged blocks) rather than short motifs. This predictive model can be used to identify potentially important residues in other proteins, improving our understanding of their functions. The trained model can be run directly from the Jupyter Notebook in the GitHub repository using Binder (mybinder.org). The only required input is the primary sequence. The training scripts are available on GitHub (https://github.com/allmwh/IFF). The training datasets have been deposited in an Open Science Framework repository (https://osf.io/jk29b).

Sequence-specific mapping of the interaction between urea and unfolded ubiquitin from ensemble analysis of NMR and small angle scattering data.

The molecular details of how urea interacts with, and eventually denatures proteins, remain largely unknown. In this study we have used extensive experimental NMR data, in combination with statistical coil ensemble modeling and small-angle scattering, to analyze the conformational behavior of the protein ubiquitin in the presence of urea. In order to develop an atomic resolution understanding of the denatured state, conformational ensembles of full-atom descriptions of unfolded proteins, including side chain conformations derived from rotamer libraries, are combined with random sampling of explicit urea molecules in interaction with the protein. Using this description of the conformational equilibrium, we demonstrate that the direct-binding model of urea to the protein backbone is compatible with available experimental data. We find that, in the presence of 8 M urea, between 30 and 40% of the backbone peptide groups bind a urea molecule, independently reproducing results from a model-free analysis of small-angle neutron and X-ray scattering data. Crucially, this analysis also provides sequence specific details of the interaction between urea and the protein backbone. The pattern of urea-binding along the amino-acid sequence reveals a higher level of binding in the central part of the protein, a trend which resembles independent results derived from chemical shift mapping of the urea-protein interaction. Together these results substantiate the direct-binding model and provide a framework for studying the physical basis of interactions between proteins and solvent molecules.

Mapping the potential energy landscape of intrinsically disordered proteins at amino acid resolution.

Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology. New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins. Although dependences of some NMR parameters such as chemical shifts (CSs) or residual dipolar couplings (RDCs) on structural propensity are known, so that sampling regimes are often inferred from experimental observation, there is currently no framework that allows for a statistical mapping of the available Ramachandran space of each amino acid in terms of conformational propensity. In this study we develop such an approach, combining highly efficient conformational sampling with ensemble selection to map the backbone conformational sampling of IDPs on a residue specific level. By systematically analyzing the ability of NMR data to map the conformational landscape of disordered proteins, we identify combinations of RDCs and CSs that can be used to raise conformational degeneracies inherent to different data types, and apply these approaches to characterize the conformational behavior of two intrinsically disordered proteins, the K18 domain from Tau protein and N(TAIL) from measles virus nucleoprotein. In both cases, we identify the enhanced populations of turn and helical regions in key regions of the proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling.

Modulation of structure and dynamics by disulfide bond formation in unfolded states.

During oxidative folding, the formation of disulfide bonds has profound effects on guiding the protein folding pathway. Until now, comparatively little is known about the changes in the conformational dynamics in folding intermediates of proteins that contain only a subset of their native disulfide bonds. In this comprehensive study, we probe the conformational landscape of non-native states of lysozyme containing a single native disulfide bond utilizing nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), circular dichroism (CD) data, and modeling approaches. The impact on conformational dynamics varies widely depending on the loop size of the single disulfide variants and deviates significantly from random coil predictions for both NMR and SAXS data. From these experiments, we conclude that the introduction of single disulfides spanning a large portion of the polypeptide chain shifts the structure and dynamics of hydrophobic core residues of the protein so that these regions exhibit levels of order comparable to the native state on the nanosecond time scale.

Residual dipolar couplings measured in unfolded proteins are sensitive to amino-acid-specific geometries as well as local conformational sampling.

Many functional proteins do not have well defined folded structures. In recent years, both experimental and computational approaches have been developed to study the conformational behaviour of this type of protein. It has been shown previously that experimental RDCs (residual dipolar couplings) can be used to study the backbone sampling of disordered proteins in some detail. In these studies, the backbone structure was modelled using a common geometry for all amino acids. In the present paper, we demonstrate that experimental RDCs are also sensitive to the specific geometry of each amino acid as defined by energy-minimized internal co-ordinates. We have modified the FM (flexible-Meccano) algorithm that constructs conformational ensembles on the basis of a statistical coil model, to account for these differences. The modified algorithm inherits the advantages of the FM algorithm to efficiently sample the potential energy landscape for coil conformations. The specific geometries incorporated in the new algorithm result in a better reproduction of experimental RDCs and are generally applicable for further studies to characterize the conformational properties of intrinsically disordered proteins. In addition, the internal-co-ordinate-based algorithm is an order of magnitude more efficient, and facilitates side-chain construction, surface osmolyte simulation, spin-label distribution sampling and proline cis/trans isomer simulation.

The return of the rings: Evolutionary convergence of aromatic residues in the intrinsically disordered regions of RNA-binding proteins for liquid-liquid phase separation.

Aromatic residues appeared relatively late in the evolution of protein sequences to stabilize the globular proteins' folding core and are less in the intrinsically disordered regions (IDRs). Recent advances in protein liquid-liquid phase separation (LLPS) studies have also shown that aromatic residues in IDRs often act as "stickers" to promote multivalent interactions in forming higher-order oligomers. To study how general these structure-promoting residues are in IDRs, we compared levels of sequence disorder in RNA binding proteins (RBPs), which are often found to undergo LLPS, and the human proteome. We found that aromatic residues appear more frequently than expected in the IDRs of RBPs and, through multiple sequence alignment analysis, those aromatic residues are often conserved among chordates. Using TDP-43, FUS, and some other well-studied LLPS proteins as examples, the conserved aromatic residues are important to their LLPS-related functions. These analyses suggest that aromatic residues may have contributed twice to evolution: stabilizing structured proteins and assembling biomolecular condensates.

Phase separation driven by interchangeable properties in the intrinsically disordered regions of protein paralogs.

Paralogs, arising from gene duplications, increase the functional diversity of proteins. Protein functions in paralog families have been extensively studied, but little is known about the roles that intrinsically disordered regions (IDRs) play in their paralogs. Without a folded structure to restrain them, IDRs mutate more diversely along with evolution. However, how the diversity of IDRs in a paralog family affects their functions is unexplored. Using the RNA-binding protein Musashi family as an example, we applied multiple structural techniques and phylogenetic analysis to show how members in a paralog family have evolved their IDRs to different physicochemical properties but converge to the same function. In this example, the lower prion-like tendency of Musashi-1's IDRs, rather than Musashi-2's, is compensated by its higher α-helical propensity to assist their assembly. Our work suggests that, no matter how diverse they become, IDRs could evolve different traits to a converged function, such as liquid-liquid phase separation.