Phase transition of RNA-protein complexes into ordered hollow condensates.

Liquid-liquid phase separation of multivalent intrinsically disordered protein-RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA-protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA-protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA-protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein-RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein-RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein-RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.

Ligand interactions and the protein order-disorder energetic continuum.

Intrinsically disordered proteins as computationally predicted account for ∼1/3 of eukaryotic proteomes, are involved in a plethora of biological functions, and have been linked to several human diseases as a result of their dysfunctions. Here, we present a picture wherein an energetic continuum describes protein structural and conformational propensities, ranging from the hyperstable folded proteins on one end to the hyperdestabilized and sometimes functionally disordered proteins on the other. We distinguish between proteins that are folding-competent but disordered because of marginal stability and those that are disordered due mainly to the absence of folding code-completing structure-determining interactions, and postulate that disordered proteins that are unstructured by way of partial population of protein denatured states represent a sizable proportion of the proteome.

Structure of an EIIC sugar transporter trapped in an inward-facing conformation.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) transports sugar into bacteria and phosphorylates the sugar for metabolic consumption. The PTS is important for the survival of bacteria and thus a potential target for antibiotics, but its mechanism of sugar uptake and phosphorylation remains unclear. The PTS is composed of multiple proteins, and the membrane-embedded Enzyme IIC (EIIC) component transports sugars across the membrane. Crystal structures of two members of the glucose superfamily of EIICs, bcChbC and bcMalT, were solved in the inward-facing and outward-facing conformations, and the structures suggest that sugar translocation could be achieved by movement of a structured domain that contains the sugar-binding site. However, different conformations have not been captured on the same transporter to allow precise description of the conformational changes. Here we present a crystal structure of bcMalT trapped in an inward-facing conformation by a mercury ion that bridges two strategically placed cysteine residues. The structure allows direct comparison of the outward- and inward-facing conformations and reveals a large rigid-body motion of the sugar-binding domain and other conformational changes that accompany the rigid-body motion. All-atom molecular dynamics simulations show that the inward-facing structure is stable with or without the cross-linking. The conformational changes were further validated by single-molecule Föster resonance energy transfer (smFRET). Combined, these results establish the elevator-type mechanism of transport in the glucose superfamily of EIIC transporters.

Influenza A Virus Protein NS1 Exhibits Strain-Independent Conformational Plasticity.

Influenza A virus (IAV) nonstructural protein 1 (NS1), a potent antagonist of the host immune response, is capable of interacting with RNA and a wide range of cellular proteins. NS1 consists of an RNA-binding domain (RBD) and an effector domain (ED) separated by a flexible linker region (LR). H5N1-NS1 has a characteristic 5-residue deletion in the LR, with either G (minor group) or E (major group) at the 71st position, and non-H5N1-NS1 contains E71 with an intact linker. Based on the orientation of the ED with respect to the RBD, previous crystallographic studies have shown that minor group H5N1-NS1(G71), a non-H5N1-NS1 [H6N6-NS1(E71)], and the LR deletion mutant H6N6-NS1(Δ80-84/E71) mimicking the major group H5N1-NS1 exhibit "open," "semiopen," and "closed" conformations, respectively, suggesting that NS1 exhibits a strain-dependent conformational preference. Here we report the first crystal structure of a naturally occurring H5N1-NS1(E71) and show that it adopts an open conformation similar to that of the minor group of H5N1-NS1 [H5N1-NS1(G71)]. We also show that H6N6-NS1(Δ80-84/E71) under a different crystallization condition and H6N6-NS1(Δ80-84/G71) also exhibit open conformations, suggesting that NS1 can adopt an open conformation irrespective of E or G at the 71st position. Our single-molecule fluorescence resonance energy transfer (FRET) analysis to investigate the conformational preference of NS1 in solution showed that all NS1 constructs predominantly exist in an open conformation. Further, our coimmunoprecipitation and binding studies showed that they all bind to cellular factors with similar affinities. Taken together, our studies suggest that NS1 exhibits strain-independent structural plasticity that allows it to interact with a wide variety of cellular ligands during viral infection.IMPORTANCE IAV is responsible for several pandemics over the last century and continues to infect millions annually. The frequent rise in drug-resistant strains necessitates exploring novel targets for developing antiviral drugs that can reduce the global burden of influenza infection. Because of its critical role in the replication and pathogenesis of IAV, nonstructural protein 1 (NS1) is a potential target for developing antivirals. Previous studies suggested that NS1 adopts strain-dependent "open," "semiopen," and "closed" conformations. Here we show, based on three crystal structures, that NS1 irrespective of strain differences can adopt an open conformation. We further show that NS1 from different strains primarily exists in an open conformation in solution and binds to cellular proteins with a similar affinity. Together, our findings suggest that conformational polymorphism facilitated by a flexible linker is intrinsic to NS1, and this may be the underlying factor allowing NS1 to bind several cellular factors during IAV replication.

Interplay between Short-Range Attraction and Long-Range Repulsion Controls Reentrant Liquid Condensation of Ribonucleoprotein-RNA Complexes.

In eukaryotic cells, ribonucleoproteins (RNPs) form mesoscale condensates by liquid-liquid phase separation that play essential roles in subcellular dynamic compartmentalization. The formation and dissolution of many RNP condensates are finely dependent on the RNA-to-RNP ratio, giving rise to a windowlike phase separation behavior. This is commonly referred to as reentrant liquid condensation (RLC). Here, using ribonucleoprotein-inspired polypeptides with low-complexity RNA-binding sequences as well as an archetypal disordered RNP, fused in sarcoma, as model systems, we investigate the molecular driving forces underlying this nonmonotonous phase transition. We show that an interplay between short-range cation-π attractions and long-range electrostatic forces governs the heterotypic RLC behavior of RNP-RNA complexes. Short-range attractions, which can be encoded by both polypeptide chain primary sequence and nucleic acid base sequence, control the two-phase coexistence regime, regulate material properties of polypeptide-RNA condensates, and oppose condensate reentrant dissolution. In the presence of excess RNA, a competition between short-range attraction and long-range electrostatic repulsion drives the formation of a colloidlike cluster phase. With increasing short-range attraction, the fluid dynamics of the cluster phase is arrested, leading to the formation of a colloidal gel. Our results reveal that phase behavior, supramolecular organization, and material states of RNP-RNA assemblies are controlled by a dynamic interplay between molecular interactions at different length scales.

A Chemical Chaperone Decouples TDP-43 Disordered Domain Phase Separation from Fibrillation.

Ribonucleoprotein (RNP) condensations through liquid-liquid phase separation play vital roles in the dynamic formation-dissolution of stress granules (SGs). These condensations are, however, usually assumed to be linked to pathologic fibrillation. Here, we show that physiologic condensation and pathologic fibrillation of RNPs are independent processes that can be unlinked with the chemical chaperone trimethylamine N-oxide (TMAO). Using the low-complexity disordered domain of the archetypical SG-protein TDP-43 as a model system, we show that TMAO enhances RNP liquid condensation yet inhibits protein fibrillation. Our results demonstrate effective decoupling of physiologic condensation from pathologic aggregation and suggest that selective targeting of protein fibrillation (without altering condensation) can be employed as a therapeutic strategy for RNP aggregation-associated degenerative disorders.

Denaturant-specific effects on the structural energetics of a protein-denatured ensemble.

Protein thermodynamic stability is intricately linked to cellular function, and altered stability can lead to dysfunction and disease. The linear extrapolation model (LEM) is commonly used to obtain protein unfolding free energies ([Formula: see text]) by extrapolation of solvent denaturation data to zero denaturant concentration. However, for some proteins, different denaturants result in non-coincident LEM-derived [Formula: see text] values, raising questions about the inherent assumption that the obtained [Formula: see text] values are intrinsic to the protein. Here, we used single-molecule FRET measurements to better understand such discrepancies by directly probing changes in the dimensions of the protein G B1 domain (GB1), a well-studied protein folding model, upon urea and guanidine hydrochloride denaturation. A comparison of the results for the two denaturants suggests denaturant-specific structural energetics in the GB1 denatured ensemble, revealing a role of the denatured state in the variable thermodynamic behavior of proteins.

Direct Single-Molecule Observation of Sequential DNA Bending Transitions by the Sox2 HMG Box.

Sox2 is a pioneer transcription factor that initiates cell fate reprogramming through locus-specific differential regulation. Mechanistically, it was assumed that Sox2 achieves its regulatory diversity via heterodimerization with partner transcription factors. Here, utilizing single-molecule fluorescence spectroscopy, we show that Sox2 alone can modulate DNA structural landscape in a dosage-dependent manner. We propose that such stoichiometric tuning of regulatory DNAs is crucial to the diverse biological functions of Sox2, and represents a generic mechanism of conferring functional plasticity and multiplicity to transcription factors.

Reentrant Phase Transition Drives Dynamic Substructure Formation in Ribonucleoprotein Droplets.

Intracellular ribonucleoprotein (RNP) granules are membrane-less droplet organelles that are thought to regulate posttranscriptional gene expression. While liquid-liquid phase separation may drive RNP granule assembly, the mechanisms underlying their supramolecular dynamics and internal organization remain poorly understood. Herein, we demonstrate that RNA, a primary component of RNP granules, can modulate the phase behavior of RNPs by controlling both droplet assembly and dissolution in vitro. Monotonically increasing the RNA concentration initially leads to droplet assembly by complex coacervation and subsequently triggers an RNP charge inversion, which promotes disassembly. This RNA-mediated reentrant phase transition can drive the formation of dynamic droplet substructures (vacuoles) with tunable lifetimes. We propose that active cellular processes that can create an influx of RNA into RNP granules, such as transcription, can spatiotemporally control the organization and dynamics of such liquid-like organelles.

The N-Terminal Domain of ALS-Linked TDP-43 Assembles without Misfolding.

Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) misfolding is implicated in several neurodegenerative diseases characterized by aggregated protein inclusions. Misfolding is believed to be mediated by both the N- and C-terminus of TDP-43; however, the mechanistic basis of the contribution of individual domains in the process remained elusive. Here, using single-molecule fluorescence and ensemble biophysical techniques, and a wide range of pH and temperature conditions, we show that TDP-43NTD is thermodynamically stable, well-folded and undergoes reversible oligomerization. We propose that, in full-length TDP-43, association between folded N-terminal domains enhances the propensity of the intrinsically unfolded C-terminal domains to drive pathological aggregation.

Two-Dimensional Crowding Uncovers a Hidden Conformation of α-Synuclein.

The intrinsically disordered protein (IDP), α-synuclein (αS), is well-known for phospholipid membrane binding-coupled folding into tunable helical conformers. Here, using single-molecule experiments in conjunction with ensemble assays and a theoretical model, we present a unique case demonstrating that the interaction-folding landscape of αS can be tuned by two-dimensional (2D) crowding through simultaneous binding of a second protein on the bilayer surface. Unexpectedly, the experimental data show a clear deviation from a simple competitive inhibition model, but are consistent with a bimodal inhibition mechanism wherein membrane binding of a second protein (a membrane interacting chaperone, Hsp27, in this case) differentially inhibits two distinct modules of αS-membrane interaction. As a consequence, αS molecules are forced to access a hidden conformational state on the phospholipid bilayer in which only the higher-affinity module remains membrane-bound. Our results demonstrate that macromolecular crowding in two dimensions can play a significant role in shaping the conformational landscape of membrane-binding IDPs with multiple binding modes.

Forced folding of a disordered protein accesses an alternative folding landscape.

Intrinsically disordered proteins (IDPs) are involved in diverse cellular functions. Many IDPs can interact with multiple binding partners, resulting in their folding into alternative ligand-specific functional structures. For such multi-structural IDPs, a key question is whether these multiple structures are fully encoded in the protein sequence, as is the case in many globular proteins. To answer this question, here we employed a combination of single-molecule and ensemble techniques to compare ligand-induced and osmolyte-forced folding of α-synuclein. Our results reveal context-dependent modulation of the protein's folding landscape, suggesting that the codes for the protein's native folds are partially encoded in its primary sequence, and are completed only upon interaction with binding partners. Our findings suggest a critical role for cellular interactions in expanding the repertoire of folds and functions available to disordered proteins.

Counteracting chemical chaperone effects on the single-molecule α-synuclein structural landscape.

Protein structure and function depend on a close interplay between intrinsic folding energy landscapes and the chemistry of the protein environment. Osmolytes are small-molecule compounds that can act as chemical chaperones by altering the environment in a cellular context. Despite their importance, detailed studies on the role of these chemical chaperones in modulating structure and dimensions of intrinsically disordered proteins have been limited. Here, we used single-molecule Förster resonance energy transfer to test the counteraction hypothesis of counterbalancing effects between the protecting osmolyte trimethylamine-N-oxide (TMAO) and denaturing osmolyte urea for the case of α-synuclein, a Parkinson's disease-linked protein whose monomer exhibits significant disorder. The single-molecule experiments, which avoid complications from protein aggregation, do not exhibit clear solvent-induced cooperative protein transitions for these osmolytes, unlike results from previous studies on globular proteins. Our data demonstrate the ability of TMAO and urea to shift α-synuclein structures towards either more compact or expanded average dimensions. Strikingly, the experiments directly reveal that a 21 [urea][TMAO] ratio has a net neutral effect on the protein's dimensions, a result that holds regardless of the absolute osmolyte concentrations. Our findings shed light on a surprisingly simple aspect of the interplay between urea and TMAO on α-synuclein in the context of intrinsically disordered proteins, with potential implications for the biological roles of such chemical chaperones. The results also highlight the strengths of single-molecule experiments in directly probing the chemical physics of protein structure and disorder in more chemically complex environments.

Heterotypic interactions can drive selective co-condensation of prion-like low-complexity domains of FET proteins and mammalian SWI/SNF complex.

Prion-like domains (PLDs) are low-complexity protein sequences enriched within nucleic acid-binding proteins including those involved in transcription and RNA processing. PLDs of FUS and EWSR1 play key roles in recruiting chromatin remodeler mammalian SWI/SNF (mSWI/SNF) complex to oncogenic FET fusion protein condensates. Here, we show that disordered low-complexity domains of multiple SWI/SNF subunits are prion-like with a strong propensity to undergo intracellular phase separation. These PLDs engage in sequence-specific heterotypic interactions with the PLD of FUS in the dilute phase at sub-saturation conditions, leading to the formation of PLD co-condensates. In the dense phase, homotypic and heterotypic PLD interactions are highly cooperative, resulting in the co-mixing of individual PLD phases and forming spatially homogeneous condensates. Heterotypic PLD-mediated positive cooperativity in protein-protein interaction networks is likely to play key roles in the co-phase separation of mSWI/SNF complex with transcription factors containing homologous low-complexity domains.

Heterotypic interactions in the dilute phase can drive co-condensation of prion-like low-complexity domains of FET proteins and mammalian SWI/SNF complex.

Prion-like domains (PLDs) are low-complexity protein sequences enriched within nucleic acid-binding proteins including those involved in transcription and RNA processing. PLDs of FUS and EWSR1 play key roles in recruiting chromatin remodeler mammalian SWI/SNF complex to oncogenic FET fusion protein condensates. Here, we show that disordered low-complexity domains of multiple SWI/SNF subunits are prion-like with a strong propensity to undergo intracellular phase separation. These PLDs engage in sequence-specific heterotypic interactions with the PLD of FUS in the dilute phase at sub-saturation conditions, leading to the formation of PLD co-condensates. In the dense phase, homotypic and heterotypic PLD interactions are highly cooperative, resulting in the co-mixing of individual PLD phases and forming spatially homogeneous co-condensates. Heterotypic PLD-mediated positive cooperativity in protein-protein interaction networks is likely to play key roles in the co-phase separation of mSWI/SNF complex with transcription factors containing homologous low-complexity domains.

Ectopic biomolecular phase transitions: fusion proteins in cancer pathologies.

Biomolecular condensates are membraneless organelles (MLOs) that are enriched in specific proteins and nucleic acids, compartmentalized to perform biochemical functions. Such condensates are formed by phase separation (PS) enabled by protein domains that allow multivalent interactions. Chromosomal translocation-derived in-frame gene fusions often generate proteins with non-native domain combinations that rewire protein-protein interaction networks. Several recent studies have shown that, for a subset of these fusion proteins, pathogenesis can be driven by the ability of the fusion protein to undergo phase transitions at non-physiological cellular locations to form ectopic condensates. We highlight how such ectopic phase transitions can alter biological processes and posit that dysfunction via protein PS at non-physiological locations represents a generic route to oncogenic transformation.

FUS oncofusion protein condensates recruit mSWI/SNF chromatin remodeler via heterotypic interactions between prion-like domains.

Fusion transcription factors generated by genomic translocations are common drivers of several types of cancers including sarcomas and leukemias. Oncofusions of the FET (FUS, EWSR1, and TAF15) family proteins result from the fusion of the prion-like domain (PLD) of FET proteins to the DNA-binding domain (DBD) of certain transcription regulators and are implicated in aberrant transcriptional programs through interactions with chromatin remodelers. Here, we show that FUS-DDIT3, a FET oncofusion protein, undergoes PLD-mediated phase separation into liquid-like condensates. Nuclear FUS-DDIT3 condensates can recruit essential components of the global transcriptional machinery such as the chromatin remodeler SWI/SNF. The recruitment of mammalian SWI/SNF (mSWI/SNF) is driven by heterotypic PLD-PLD interactions between FUS-DDIT3 and core subunits of SWI/SNF, such as the catalytic component BRG1. Further experiments with single-molecule correlative force-fluorescence microscopy support a model wherein the fusion protein forms condensates on DNA surface and enrich BRG1 to activate transcription by ectopic chromatin remodeling. Similar PLD-driven co-condensation of mSWI/SNF with transcription factors can be employed by other oncogenic fusion proteins with a generic PLD-DBD domain architecture for global transcriptional reprogramming.

Programmable viscoelasticity in protein-RNA condensates with disordered sticker-spacer polypeptides.

Liquid-liquid phase separation of multivalent proteins and RNAs drives the formation of biomolecular condensates that facilitate membrane-free compartmentalization of subcellular processes. With recent advances, it is becoming increasingly clear that biomolecular condensates are network fluids with time-dependent material properties. Here, employing microrheology with optical tweezers, we reveal molecular determinants that govern the viscoelastic behavior of condensates formed by multivalent Arg/Gly-rich sticker-spacer polypeptides and RNA. These condensates behave as Maxwell fluids with an elastically-dominant rheological response at shorter timescales and a liquid-like behavior at longer timescales. The viscous and elastic regimes of these condensates can be tuned by the polypeptide and RNA sequences as well as their mixture compositions. Our results establish a quantitative link between the sequence- and structure-encoded biomolecular interactions at the microscopic scale and the rheological properties of the resulting condensates at the mesoscale, enabling a route to systematically probe and rationally engineer biomolecular condensates with programmable mechanics.

Single-Molecule FRET Detection of Early-Stage Conformations in α-Synuclein Aggregation.

Misfolding and aggregation of α-synuclein are linked to many neurodegenerative disorders, including Parkinson's and Alzheimer's disease. Despite intense research efforts, detailed structural characterization of early conformational transitions that initiate and drive α-synuclein aggregation remains elusive often due to the low sensitivity and ensemble averaging of commonly used techniques. Single-molecule Förster resonance energy transfer (smFRET) provides unique advantages in detecting minor conformations that initiate protein pathologic aggregation. In this chapter, we describe an smFRET-based method for characterizing early conformational conversions that are responsible for α-synuclein self-assembly and aggregation.