Structural details of helix-mediated TDP-43 C-terminal domain multimerization.

The primarily disordered C-terminal domain (CTD) of TAR DNA binding protein-43 (TDP-43), a key nuclear protein in RNA metabolism, forms neuronal inclusions in several neurodegenerative diseases. A conserved region (CR, spanning residues 319-341) in CTD forms transient helix-helix contacts important for its higher-order oligomerization and function that are disrupted by ALS-associated mutations. However, the structural details of CR assembly and the explanation for several ALS-associated variants' impact on phase separation and function remain unclear due to challenges in analyzing the dynamic association of TDP-43 CTD using traditional structural biology approaches. By employing an integrative approach, combining biophysical experiments, biochemical assays, AlphaFold2-Multimer (AF2-Multimer), and atomistic simulations, we generated structural models of helical oligomerization of TDP-43 CR. Using NMR, we first established that the native state of TDP-43 CR under physiological conditions is α-helical. Next, alanine scanning mutagenesis revealed that while hydrophobic residues in the CR are important for CR assembly, phase separation and TDP-43 nuclear retention function, polar residues down regulate these processes. Finally, pairing AF2-Multimer modeling with AAMD simulations indicated that dynamic, oligomeric assemblies of TDP-43 that are stabilized by a methionine-rich core with specific contributions from a tryptophan/leucine pair. In conclusion, our results advance the structural understanding of the mechanisms driving TDP-43 function and provide a window into the initial stages of its conversion into pathogenic aggregates.

Intra-condensate demixing of TDP-43 inside stress granules generates pathological aggregates.

Cytosolic aggregation of the nuclear protein TDP-43 is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43 enriched phase within stress granules, which subsequently transitions into pathological aggregates. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We conclude that up-concentration inside condensates and simultaneous exposure to environmental stress could be a general pathway for protein aggregation, with intra-condensate demixing constituting a key intermediate step.

Structure and interactions of prion-like domains in transcription factor Efg1 phase separation.

Candida albicans, a prominent member of the human microbiome, can make an opportunistic switch from commensal coexistence to pathogenicity accompanied by an epigenetic shift between the white and opaque cell states. This transcriptional switch is under precise regulation by a set of transcription factors (TFs), with Enhanced Filamentous Growth Protein 1 (Efg1) playing a central role. Previous research has emphasized the importance of Efg1's prion-like domain (PrLD) and the protein's ability to undergo phase separation for the white-to-opaque transition of C. albicans. However, the underlying molecular mechanisms of Efg1 phase separation have remained underexplored. In this study, we delved into the biophysical basis of Efg1 phase separation, revealing the significant contribution of both N-terminal (N) and C-terminal (C) PrLDs. Through NMR structural analysis, we found that Efg1 N-PrLD and C-PrLD are mostly disordered but have prominent partial α-helical secondary structures in both domains. NMR titration experiments suggest that the partially helical structures in N-PrLD act as hubs for self-interaction as well as Efg1 interaction with RNA. Using condensed-phase NMR spectroscopy, we uncovered diverse amino acid interactions underlying Efg1 phase separation. Particularly, we highlight the indispensable role of tyrosine residues within the transient α-helical structures of PrLDs particularly in the N-PrLD compared to the C-PrLD in stabilizing phase separation. Our study provides evidence that the transient α-helical structure is present in the phase-separated state and highlights the particular importance of aromatic residues within these structures for phase separation. Together, these results enhance the understanding of C. albicans transcription factor interactions that lead to virulence and provide a crucial foundation for potential antifungal therapies targeting the transcriptional switch.

Expanding the molecular grammar of polar residues and arginine in FUS prion-like domain phase separation and aggregation.

A molecular grammar governing low-complexity prion-like domains phase separation (PS) has been proposed based on mutagenesis experiments that identified tyrosine and arginine as primary drivers of phase separation via aromatic-aromatic and aromatic-arginine interactions. Here we show that additional residues make direct favorable contacts that contribute to phase separation, highlighting the need to account for these contributions in PS theories and models. We find that tyrosine and arginine make important contacts beyond only tyrosine-tyrosine and tyrosine-arginine, including arginine-arginine contacts. Among polar residues, glutamine in particular contributes to phase separation with sequence/position-specificity, making contacts with both tyrosine and arginine as well as other residues, both before phase separation and in condensed phases. For glycine, its flexibility, not its small solvation volume, favors phase separation by allowing favorable contacts between other residues and inhibits the liquid-to-solid (LST) transition. Polar residue types also make sequence-specific contributions to aggregation that go beyond simple rules, which for serine positions is linked to formation of an amyloid-core structure by the FUS low-complexity domain. Hence, here we propose a revised molecular grammar expanding the role of arginine and polar residues in prion-like domain protein phase separation and aggregation.

Dual DNA/RNA-binding factor regulates dynamics of hnRNP splicing condensates.

Despite decades of research, mechanisms by which co-transcriptional alternative splicing events are targeted to the correct genomic locations to drive cell fate decisions remain unknown. By combining structural and molecular approaches, we define a new mechanism by which an essential transcription factor (TF) targets co-transcriptional splicing through physical and functional interaction with RNA and RNA binding proteins (RBPs). We show that an essential TF co-transcriptionally regulates sex-specific alternative splicing by directly interacting with a subset of target RNAs on chromatin and modulating the dynamics of hnRNPA2 homolog nuclear splicing condensates.

A complex network of interdomain interactions underlies the conformational ensemble of monomeric TDP-43 and modulates its phase behavior.

TAR DNA-binding protein 43 (TDP-43) is a multidomain protein involved in the regulation of RNA metabolism, and its aggregates have been observed in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Numerous studies indicate TDP-43 can undergo liquid-liquid phase separation (LLPS) in vitro and is a component of biological condensates. Homo-oligomerization via the folded N-terminal domain (aa:1-77) and the conserved helical region (aa:319-341) of the disordered, C-terminal domain is found to be an important driver of TDP-43 phase separation. However, a comprehensive molecular view of TDP-43 phase separation, particularly regarding the nature of heterodomain interactions, is lacking due to the challenges associated with its stability and purification. Here, we utilize all-atom and coarse-grained (CG) molecular dynamics (MD) simulations to uncover the network of interdomain interactions implicated in TDP-43 phase separation. All-atom simulations uncovered the presence of transient, interdomain interactions involving flexible linkers, RNA-recognition motif (RRM) domains and a charged segment of disordered C-terminal domain (CTD). CG simulations indicate these inter-domain interactions which affect the conformational landscape of TDP-43 in the dilute phase are also prevalent in the condensed phase. Finally, sequence and surface charge distribution analysis coupled with all-atom simulations (at high salt) confirmed that the transient interdomain contacts are predominantly electrostatic in nature. Overall, our findings from multiscale simulations lead to a greater appreciation of the complex interaction network underlying the structural landscape and phase separation of TDP-43.

Liquid-Liquid Phase Separation of the Intrinsically Disordered Domain of the Fused in Sarcoma Protein Results in Substantial Slowing of Hydration Dynamics.

Formation of liquid condensates plays a critical role in biology via localization of different components or via altered hydrodynamic transport, yet the hydrogen-bonding environment within condensates, pivotal for solvation, has remained elusive. We explore the hydrogen-bond dynamics within condensates formed by the low-complexity domain of the fused in sarcoma protein. Probing the hydrogen-bond dynamics sensed by condensate proteins using two-dimensional infrared spectroscopy of the protein amide I vibrations, we find that frequency-frequency correlations of the amide I vibration decay on a picosecond time scale. Interestingly, these dynamics are markedly slower for proteins in the condensate than in a homogeneous protein solution, indicative of different hydration dynamics. All-atom molecular dynamics simulations confirm that lifetimes of hydrogen-bonds between water and the protein are longer in the condensates than in the protein in solution. Altered hydrogen-bonding dynamics may contribute to unique solvation and reaction dynamics in such condensates.

Structure and position-specific interactions of prion-like domains in transcription factor Efg1 phase separation.

Candida albicans, a prominent member of the human microbiome, can make an opportunistic switch from commensal coexistence to pathogenicity accompanied by an epigenetic shift between the white and opaque cell states. This transcriptional switch is under precise regulation by a set of transcription factors (TFs), with Enhanced Filamentous Growth Protein 1 (Efg1) playing a central role. Previous research has emphasized the importance of Egf1's prion-like domain (PrLD) and the protein's ability to undergo phase separation for the white-to-opaque transition of C. albicans. However, the underlying molecular mechanisms of Efg1 phase separation have remained underexplored. In this study, we delved into the biophysical basis of Efg1 phase separation, revealing the significant contribution of both N-terminal (N) and C-terminal (C) PrLDs. Through NMR structural analysis, we found that Efg1 N-PrLD and C-PrLD are mostly disordered though have prominent partial α-helical secondary structures in both domains. NMR titration experiments suggest that the partially helical structures in N-PrLD act as hubs for self-interaction as well as Efg1 interaction with RNA. Using condensed-phase NMR spectroscopy, we uncovered diverse amino acid interactions underlying Efg1 phase separation. Particularly, we highlight the indispensable role of tyrosine residues within the transient α-helical structures of PrLDs particularly in the N-PrLD compared to the C-PrLD in stabilizing phase separation. Our study provides evidence that the transient α-helical structure is present in the phase separated state and highlights the particular importance of aromatic residues within these structures for phase separation. Together, these results enhance the understanding of C. albicans TF interactions that lead to virulence and provide a crucial foundation for potential antifungal therapies targeting the transcriptional switch.

Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in Xenopus oocytes.

Proteins containing both intrinsically disordered regions (IDRs) and RNA binding domains (RBDs) can phase separate in vitro, forming bodies similar to cellular biomolecular condensates. However, how IDR and RBD domains contribute to in vivo recruitment of proteins to biomolecular condensates remains poorly understood. Here, we analyzed the roles of IDRs and RBDs in L-bodies, biomolecular condensates present in Xenopus oocytes. We show that a cytoplasmic isoform of hnRNPAB, which contains two RBDs and an IDR, is highly enriched in L-bodies. While both of these domains contribute to hnRNPAB self-association and phase separation in vitro and mediate enrichment into L-bodies in oocytes, neither the RBDs nor the IDR replicate the localization of full-length hnRNPAB. Our results suggest a model where the additive effects of the IDR and RBDs regulate hnRNPAB partitioning into L-bodies. This model likely has widespread applications as proteins containing RBD and IDR domains are common biomolecular condensate residents.

Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains.

RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and human motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders.

A synergy between site-specific and transient interactions drives the phase separation of a disordered, low-complexity domain.

TAR DNA-binding protein 43 (TDP-43) is involved in key processes in RNA metabolism and is frequently implicated in many neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. The prion-like, disordered C-terminal domain (CTD) of TDP-43 is aggregation-prone, can undergo liquid-liquid phase separation (LLPS) in isolation, and is critical for phase separation (PS) of the full-length protein under physiological conditions. While a short conserved helical region (CR, spanning residues 319-341) promotes oligomerization and is essential for LLPS, aromatic residues in the flanking disordered regions (QN-rich, IDR1/2) are also found to play a critical role in PS and aggregation. Compared with other phase-separating proteins, TDP-43 CTD has a notably distinct sequence composition including many aliphatic residues such as methionine and leucine. Aliphatic residues were previously suggested to modulate the apparent viscosity of the resulting phases, but their direct contribution toward CTD phase separation has been relatively ignored. Using multiscale simulations coupled with in vitro saturation concentration (csat) measurements, we identified the importance of aromatic residues while also suggesting an essential role for aliphatic methionine residues in promoting single-chain compaction and LLPS. Surprisingly, NMR experiments showed that transient interactions involving phenylalanine and methionine residues in the disordered flanking regions can directly enhance site-specific, CR-mediated intermolecular association. Overall, our work highlights an underappreciated mode of biomolecular recognition, wherein both transient and site-specific hydrophobic interactions act synergistically to drive the oligomerization and phase separation of a disordered, low-complexity domain.

Fibril formation and ordering of disordered FUS LC driven by hydrophobic interactions.

Biomolecular condensates, protein-rich and dynamic membrane-less organelles, play critical roles in a range of subcellular processes, including membrane trafficking and transcriptional regulation. However, aberrant phase transitions of intrinsically disordered proteins in biomolecular condensates can lead to the formation of irreversible fibrils and aggregates that are linked to neurodegenerative diseases. Despite the implications, the interactions underlying such transitions remain obscure. Here we investigate the role of hydrophobic interactions by studying the low-complexity domain of the disordered 'fused in sarcoma' (FUS) protein at the air/water interface. Using surface-specific microscopic and spectroscopic techniques, we find that a hydrophobic interface drives fibril formation and molecular ordering of FUS, resulting in solid-like film formation. This phase transition occurs at 600-fold lower FUS concentration than required for the canonical FUS low-complexity liquid droplet formation in bulk. These observations highlight the importance of hydrophobic effects for protein phase separation and suggest that interfacial properties drive distinct protein phase-separated structures.

Phase separation in fungi.

Phase separation, in which macromolecules partition into a concentrated phase that is immiscible with a dilute phase, is involved with fundamental cellular processes across the tree of life. We review the principles of phase separation and highlight how it impacts diverse processes in the fungal kingdom. These include the regulation of autophagy, cell signalling pathways, transcriptional circuits and the establishment of asymmetry in fungal cells. We describe examples of stable, phase-separated assemblies including membraneless organelles such as the nucleolus as well as transient condensates that also arise through phase separation and enable cells to rapidly and reversibly respond to important environmental cues. We showcase how research into phase separation in model yeasts, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, in conjunction with that in plant and human fungal pathogens, such as Ashbya gossypii and Candida albicans, is continuing to enrich our understanding of fundamental molecular processes.

Interactions between FUS and the C-terminal Domain of Nup62 are Sufficient for their Co-phase Separation into Amorphous Assemblies.

Deficient nucleocytoplasmic transport is emerging as a pathogenic feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), including in ALS caused by mutations in Fused in Sarcoma (FUS). Recently, both wild-type and ALS-linked mutant FUS were shown to directly interact with the phenylalanine-glycine (FG)-rich nucleoporin 62 (Nup62) protein, where FUS WT/ Nup62 interactions were enriched within the nucleus but ALS-linked mutant FUS/ Nup62 interactions were enriched within the cytoplasm of cells. Nup62 is a central channel Nup that has a prominent role in forming the selectivity filter within the nuclear pore complex and in regulating effective nucleocytoplasmic transport. Under conditions where FUS phase separates into liquid droplets in vitro, the addition of Nup62 caused the synergistic formation of amorphous assemblies containing both FUS and Nup62. Here, we examined the molecular determinants of this process using recombinant FUS and Nup62 proteins and biochemical approaches. We demonstrate that the structured C-terminal domain of Nup62 containing an alpha-helical coiled-coil region plays a dominant role in binding FUS and is sufficient for inducing the formation of FUS/Nup62 amorphous assemblies. In contrast, the natively unstructured, F/G repeat-rich N-terminal domain of Nup62 modestly contributed to FUS/Nup62 phase separation behavior. Expression of individual Nup62 domain constructs in human cells confirmed that the Nup62 C-terminal domain is essential for localization of the protein to the nuclear envelope. Our results raise the possibility that interactions between FUS and the C-terminal domain of Nup62 can influence the function of Nup62 under physiological and/or pathological conditions.

An enhancer sequence in the intrinsically disordered region of FtsZ promotes polymer-guided substrate processing by ClpXP protease.

The essential bacterial division protein in Escherichia coli, FtsZ, assembles into the FtsZ-ring at midcell and recruits other proteins to the division site to promote septation. A region of the FtsZ amino acid sequence that links the conserved polymerization domain to a C-terminal protein interaction site was predicted to be intrinsically disordered and has been implicated in modulating spacing and architectural arrangements of FtsZ filaments. While the majority of cell division proteins that directly bind to FtsZ engage either the polymerization domain or the C-terminal interaction site, ClpX, the recognition and unfolding component of the bacterial ClpXP proteasome, has a secondary interaction with the predicted intrinsically disordered region (IDR) of FtsZ when FtsZ is polymerized. Here, we use NMR spectroscopy and reconstituted degradation reactions in vitro to demonstrate that this linker region is indeed disordered in solution and, further, that amino acids in the IDR of FtsZ enhance the degradation in polymer-guided interactions.

Reversible Kinetic Trapping of FUS Biomolecular Condensates.

Formation of membrane-less organelles by self-assembly of disordered proteins can be triggered by external stimuli such as pH, salt, or temperature. These organelles, called biomolecular condensates, have traditionally been classified as liquids, gels, or solids with limited subclasses. Here, the authors show that a thermal trigger can lead to formation of at least two distinct liquid condensed phases of the fused in sarcoma low complexity (FUS LC) domain. Forming FUS LC condensates directly at low temperature leads to formation of metastable, kinetically trapped condensates that show arrested coalescence, escape from which to untrapped condensates can be achieved via thermal annealing. Using experimental and computational approaches, the authors find that molecular structure of interfacial FUS LC in kinetically trapped condensates is distinct (more ß-sheet like) compared to untrapped FUS LC condensates. Moreover, molecular motion within kinetically trapped condensates is substantially slower compared to that in untrapped condensates thereby demonstrating two unique liquid FUS condensates. Controlling condensate thermodynamic state, stability, and structure with a simple thermal switch may contribute to pathological protein aggregate stability and provides a facile method to trigger condensate mixing for biotechnology applications.

Molecular interactions contributing to FUS SYGQ LC-RGG phase separation and co-partitioning with RNA polymerase II heptads.

The RNA-binding protein FUS (Fused in Sarcoma) mediates phase separation in biomolecular condensates and functions in transcription by clustering with RNA polymerase II. Specific contact residues and interaction modes formed by FUS and the C-terminal heptad repeats of RNA polymerase II (CTD) have been suggested but not probed directly. Here we show how RGG domains contribute to phase separation with the FUS N-terminal low-complexity domain (SYGQ LC) and RNA polymerase II CTD. Using NMR spectroscopy and molecular simulations, we demonstrate that many residue types, not solely arginine-tyrosine pairs, form condensed-phase contacts via several interaction modes including, but not only sp2-π and cation-π interactions. In phases also containing RNA polymerase II CTD, many residue types form contacts, including both cation-π and hydrogen-bonding interactions formed by the conserved human CTD lysines. Hence, our data suggest a surprisingly broad array of residue types and modes explain co-phase separation of FUS and RNA polymerase II.

TDP-43 condensation properties specify its RNA-binding and regulatory repertoire.

Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.

The oncogenic transcription factor FUS-CHOP can undergo nuclear liquid-liquid phase separation.

Myxoid liposarcoma is caused by a chromosomal translocation resulting in a fusion protein comprised of the N terminus of FUS (fused in sarcoma) and the full-length transcription factor CHOP (CCAAT/enhancer-binding protein homologous protein, also known as DDIT3). FUS functions in RNA metabolism, and CHOP is a stress-induced transcription factor. The FUS-CHOP fusion protein causes unique gene expression and oncogenic transformation. Although it is clear that the FUS segment is required for oncogenic transformation, the mechanism of FUS-CHOP-induced transcriptional activation is unknown. Recently, some transcription factors and super enhancers have been proposed to undergo liquid-liquid phase separation and form membraneless compartments that recruit transcription machinery to gene promoters. Since phase separation of FUS depends on its N terminus, transcriptional activation by FUS-CHOP could result from the N terminus driving nuclear phase transitions. Here, we characterized FUS-CHOP in cells and in vitro, and observed novel phase-separating properties relative to unmodified CHOP. Our data indicate that FUS-CHOP forms phase-separated condensates that colocalize with BRD4, a marker of super enhancer condensates. We provide evidence that the FUS-CHOP phase transition is a novel oncogenic mechanism and potential therapeutic target for myxoid liposarcoma. This article has an associated First Person interview with the first author of the paper.

Interactions between ALS-linked FUS and nucleoporins are associated with defects in the nucleocytoplasmic transport pathway.

Nucleocytoplasmic transport (NCT) decline occurs with aging and neurodegeneration. Here, we investigated the NCT pathway in models of amyotrophic lateral sclerosis-fused in sarcoma (ALS-FUS). Expression of ALS-FUS led to a reduction in NCT and nucleoporin (Nup) density within the nuclear membrane of human neurons. FUS and Nups were found to interact independently of RNA in cells and to alter the phase-separation properties of each other in vitro. FUS-Nup interactions were not localized to nuclear pores, but were enriched in the nucleus of control neurons versus the cytoplasm of mutant neurons. Our data indicate that the effect of ALS-linked mutations on the cytoplasmic mislocalization of FUS, rather than on the physiochemical properties of the protein itself, underlie our reported NCT defects. An aberrant interaction between mutant FUS and Nups is underscored by studies in Drosophila, whereby reduced Nup expression rescued multiple toxic FUS-induced phenotypes, including abnormal nuclear membrane morphology in neurons.