YAP condensates are highly organized hubs.

YAP/TEAD signaling is essential for organismal development, cell proliferation, and cancer progression. As a transcriptional coactivator, how YAP activates its downstream target genes is incompletely understood. YAP forms biomolecular condensates in response to hyperosmotic stress, concentrating transcription-related factors to activate downstream target genes. However, whether YAP forms condensates under other signals, how YAP condensates organize and function, and how YAP condensates activate transcription in general are unknown. Here, we report that endogenous YAP forms sub-micron scale condensates in response to Hippo pathway regulation and actin cytoskeletal tension. YAP condensates are stabilized by the transcription factor TEAD1, and recruit BRD4, a coactivator that is enriched at active enhancers. Using single-particle tracking, we found that YAP condensates slowed YAP diffusion within condensate boundaries, a possible mechanism for promoting YAP target search. These results reveal that YAP condensate formation is a highly regulated process that is critical for YAP/TEAD target gene expression.

Disordered proteins interact with the chemical environment to tune their protective function during drying.

The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins combined with the exposure of their residues accounts for this sensitivity. One context in which IDPs play important roles that is concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family, synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results demonstrate that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet, the mechanisms underlying this synergy differ between IDP families.

Structural biases in disordered proteins are prevalent in the cell.

Intrinsically disordered proteins and protein regions (IDPs) are prevalent in all proteomes and are essential to cellular function. Unlike folded proteins, IDPs exist in an ensemble of dissimilar conformations. Despite this structural plasticity, intramolecular interactions create sequence-specific structural biases that determine an IDP ensemble's three-dimensional shape. Such structural biases can be key to IDP function and are often measured in vitro, but whether those biases are preserved inside the cell is unclear. Here we show that structural biases in IDP ensembles found in vitro are recapitulated inside human-derived cells. We further reveal that structural biases can change in a sequence-dependent manner due to changes in the intracellular milieu, subcellular localization, and intramolecular interactions with tethered well-folded domains. We propose that the structural sensitivity of IDP ensembles can be leveraged for biological function, can be the underlying cause of IDP-driven pathology or can be used to design disorder-based biosensors and actuators.

Helicity of a tardigrade disordered protein contributes to its protective function during desiccation.

To survive extreme drying (anhydrobiosis), many organisms, spanning every kingdom of life, accumulate intrinsically disordered proteins (IDPs). For decades, the ability of anhydrobiosis-related IDPs to form transient amphipathic helices has been suggested to be important for promoting desiccation tolerance. However, evidence empirically supporting the necessity and/or sufficiency of helicity in mediating anhydrobiosis is lacking. Here, we demonstrate that the linker region of CAHS D, a desiccation-related IDP from the tardigrade Hypsibius exemplaris, that contains significant helical structure, is the protective portion of this protein. Perturbing the sequence composition and grammar of the linker region of CAHS D, through the insertion of helix-breaking prolines, modulating the identity of charged residues, or replacement of hydrophobic amino acids with serine or glycine residues results in variants with different degrees of helical structure. Importantly, correlation of protective capacity and helical content in variants generated through different helix perturbing modalities does not show as strong a trend, suggesting that while helicity is important, it is not the only property that makes a protein protective during desiccation. These results provide direct evidence for the decades-old theory that helicity of desiccation-related IDPs is linked to their anhydrobiotic capacity.

Sequence-ensemble-function relationships for disordered proteins in live cells.

Intrinsically disordered protein regions (IDRs) are ubiquitous across all kingdoms of life and play a variety of essential cellular roles. IDRs exist in a collection of structurally distinct conformers known as an ensemble. An IDR's amino acid sequence determines its ensemble, which in turn can play an important role in dictating molecular function. Yet a clear link connecting IDR sequence, its ensemble properties, and its molecular function in living cells has not been directly established. Here, we set out to test this sequence-ensemble-function paradigm using a novel computational method (GOOSE) that enables the rational design of libraries of IDRs by systematically varying specific sequence properties. Using ensemble FRET, we measured the ensemble dimensions of a library of rationally designed IDRs in human-derived cell lines, revealing how IDR sequence influences ensemble dimensions in situ. Furthermore, we show that the interplay between sequence and ensemble can tune an IDR's ability to sense changes in cell volume - a de novo molecular function for these synthetic sequences. Our results establish biophysical rules for intracellular sequence-ensemble relationships, enable a new route for understanding how IDR sequences map to function in live cells, and set the ground for the design of synthetic IDRs with de novo function.

Sequence-ensemble-function relationships for disordered proteins in live cells.

Intrinsically disordered protein regions (IDRs) are ubiquitous across all kingdoms of life and play a variety of essential cellular roles. IDRs exist in a collection of structurally distinct conformers known as an ensemble. IDR amino acid sequence determines its ensemble, which in turn can play an important role in dictating molecular function. Yet a clear link connecting IDR sequence, its ensemble properties, and its molecular function in living cells has not been systematically established. Here, we set out to test this sequence-ensemble-function paradigm using a novel computational method (GOOSE) that enables the rational design of libraries of IDRs by systematically varying specific sequence properties. Using ensemble FRET, we measured the ensemble dimensions of a library of rationally designed IDRs in human-derived cell lines, revealing how IDR sequence influences ensemble dimensions in situ. Furthermore, we show that the interplay between sequence and ensemble can tune an IDR's ability to sense changes in cell volume - a de novomolecular function for these synthetic sequences. Our results establish biophysical rules for intracellular sequence-ensemble relationships, enable a new route for understanding how IDR sequences map to function in live cells, and set the ground for the design of synthetic IDRs with de novo function.

Intrinsically disordered regions are poised to act as sensors of cellular chemistry.

Intrinsically disordered proteins and protein regions (IDRs) are abundant in eukaryotic proteomes and play a wide variety of essential roles. Instead of folding into a stable structure, IDRs exist in an ensemble of interconverting conformations whose structure is biased by sequence-dependent interactions. The absence of a stable 3D structure, combined with high solvent accessibility, means that IDR conformational biases are inherently sensitive to changes in their environment. Here, we argue that IDRs are ideally poised to act as sensors and actuators of cellular physicochemistry. We review the physical principles that underlie IDR sensitivity, the molecular mechanisms that translate this sensitivity to function, and recent studies where environmental sensing by IDRs may play a key role in their downstream function.

When Phased without Water: Biophysics of Cellular Desiccation, from Biomolecules to Condensates.

The molecular machinery that enables life has evolved in water, yet many of the organisms around us are able to survive even extreme desiccation. Especially remarkable are single-cell and sedentary organisms that rely on specialized biomolecular machinery to survive in environments that are routinely subjected to a near-complete lack of water. In this review, we zoom in on the molecular level of what is happening in the cellular environment under water stress. We cover the various mechanisms by which biochemical components of the cell can dysfunction in dehydrated cells and detail the different strategies that organisms have evolved to eliminate or cope with these desiccation-induced perturbations. We specifically focus on two survival strategies: (1) the use of disordered proteins to protect the cellular environment before, during, and in the recovery from desiccation, and (2) the use of biomolecular condensates as a self-assembly mechanism that can sequester or protect specific cellular machinery in times of water stress. We provide a summary of experimental work describing the critical contributions of disordered proteins and biomolecular condensates to the cellular response to water loss and highlight their role in desiccation tolerance. Desiccation biology is an exciting area of cell biology, still far from being completely explored. Understanding it on the molecular level is bound to give us critical new insights in how life adapted/can adapt to the loss of water, spanning from the early colonization of land to how we can deal with climate change in our future.

Structural Preferences Shape the Entropic Force of Disordered Protein Ensembles.

Intrinsically disordered protein regions (IDRs) make up over 30% of the human proteome and exist in a dynamic conformational ensemble instead of a native, well-folded structure. Tethering IDRs to a surface (for example, the surface of a well-folded region of the same protein) can reduce the number of accessible conformations in these ensembles. This reduces the ensemble's conformational entropy, generating an effective entropic force that pulls away from the point of tethering. Recent experimental work has shown that this entropic force causes measurable, physiologically relevant changes to protein function. But how the magnitude of this force depends on IDR sequence remains unexplored. Here, we use all-atom simulations to analyze how structural preferences in IDR ensembles contribute to the entropic force they exert upon tethering. We show that sequence-encoded structural preferences play an important role in determining the magnitude of this force: compact, spherical ensembles generate an entropic force that can be several times higher than more extended ensembles. We further show that changes in the surrounding solution's chemistry can modulate the IDR entropic force strength. We propose that the entropic force is a sequence-dependent, environmentally tunable property of terminal IDR sequences.

TMAO: Protecting proteins from feeling the heat.

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolyte trimethylamine N-oxide (TMAO) is used by marine animals to counteract the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and urea denaturation has been extensively studied, but unlike the case of other osmolytes, the ability of TMAO to protect proteins from high temperature has not been quantified. To reveal the effect of TMAO on folded and unfolded protein ensembles and the hydration shell at different temperatures, we study a mutant of the well-characterized, fast-folding model protein B (PRB). We carried out, in total, >190 µs all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations show increased thermal stability of PRB in the presence of TMAO. Partly structured, compact ensembles are favored over the unfolded state. TMAO forms two shells near the protein: an outer shell away from the protein surface has altered H-bond lifetimes of water molecules and increases hydration of the protein to help stabilize it; a less-populated inner shell with an opposite TMAO orientation closer to the protein surface binds exclusively to basic side chains. The cooperative cosolute effect of the inner and outer shell TMAO has a small number of TMAO molecules "herding" water molecules into two hydration shells at or near the protein surface. The stabilizing effect of TMAO on our protein saturates at 1 M despite higher TMAO solubility, so there may be little evolutionary pressure for extremophiles to produce higher intracellular TMAO concentrations, if true in general.

Structural preferences shape the entropic force of disordered protein ensembles.

Intrinsically disordered protein regions (IDRs) make up over 30% of the human proteome and instead of a native, well-folded structure exist in a dynamic conformational ensemble. Tethering IDRs to a surface (for example, the surface of a well-folded region of the same protein) can reduce the number of accessible conformations in IDR ensembles. This reduces the ensemble's conformational entropy, generating an effective entropic force that pulls away from the point of tethering. Recent experimental work has shown that this entropic force causes measurable, physiologically relevant changes to protein function, but how the magnitude of this force depends on the IDR sequence remains unexplored. Here we use all-atom simulations to analyze how structural preferences encoded in dozens of IDR ensembles contribute to the entropic force they exert upon tethering. We show that sequence-encoded structural preferences play an important role in determining the magnitude of this force and that compact, spherical ensembles generate an entropic force that can be several times higher than more extended ensembles. We further show that changes in the surrounding solution's chemistry can modulate IDR entropic force strength. We propose that the entropic force is a sequence-dependent, environmentally tunable property of terminal IDR sequences.

Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells.

Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Experimental approaches for dynamically monitoring these intracellular effects are currently lacking. Here, we leverage the environmental sensitivity and structural plasticity of intrinsically disordered protein regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use as environmentally-responsive molecular tools.

A prion-like protein regulator of seed germination undergoes hydration-dependent phase separation.

Many organisms evolved strategies to survive desiccation. Plant seeds protect dehydrated embryos from various stressors and can lay dormant for millennia. Hydration is the key trigger to initiate germination, but the mechanism by which seeds sense water remains unresolved. We identified an uncharacterized Arabidopsis thaliana prion-like protein we named FLOE1, which phase separates upon hydration and allows the embryo to sense water stress. We demonstrate that biophysical states of FLOE1 condensates modulate its biological function in vivo in suppressing seed germination under unfavorable environments. We find intragenic, intraspecific, and interspecific natural variation in FLOE1 expression and phase separation and show that intragenic variation is associated with adaptive germination strategies in natural populations. This combination of molecular, organismal, and ecological studies uncovers FLOE1 as a tunable environmental sensor with direct implications for the design of drought-resistant crops, in the face of climate change.

Myomegalin regulates Hedgehog pathway by controlling PDE4D at the centrosome.

Mutations in the hedgehog (Hh) signaling are implicated in birth defects and cancers, including medulloblastoma (MB), one of the most malignant pediatric brain tumors. Current Hh inhibitors face the challenge of drug resistance and tumor relapse, urging new insights in the Hh pathway regulation. Our previous study revealed how PDE4D controls global levels of cAMP in the cytoplasm to positively regulate Hh signaling; in the present study, we found that a specific isoform PDE4D3 is tethered to the centrosome by Myomegalin (Mmg), a centrosome/Golgi-associated protein. Mmg loss dislocates PDE4D3 from the centrosome, leading to local PKA overactivation and inhibition of the Hh signaling, leaving other PKA-related pathways unaffected. Mmg loss suppresses the proliferation of granule neuron precursors and blocks the growth of MB in mouse model. Our findings specify a new regulatory mechanism of the Hh pathway and highlight an exciting therapeutic avenue for Hh-related cancers with reduced side effects.

Computational modeling of protein conformational changes - Application to the opening SARS-CoV-2 spike.

We present a new approach to compute and analyze the dynamical electro-geometric properties of proteins undergoing conformational changes. The molecular trajectory is obtained from Markov state models, and the electrostatic potential is calculated using the continuum Poisson-Boltzmann equation. The numerical electric potential is constructed using a parallel sharp numerical solver implemented on adaptive Octree grids. We introduce novel a posteriori error estimates to quantify the solution's accuracy on the molecular surface. To illustrate the approach, we consider the opening of the SARS-CoV-2 spike protein using the recent molecular trajectory simulated through the Folding@home initiative. We analyze our results, focusing on the characteristics of the receptor-binding domain and its vicinity. This work lays the foundation for a new class of hybrid computational approaches, producing high-fidelity dynamical computational measurements serving as a basis for protein bio-mechanism investigations.

Revealing the Hidden Sensitivity of Intrinsically Disordered Proteins to their Chemical Environment.

Intrinsically disordered protein-regions (IDRs) make up roughly 30% of the human proteome and are central to a wide range of biological processes. Given a lack of persistent tertiary structure, all residues in IDRs are, to some extent, solvent exposed. This extensive surface area, coupled with the absence of strong intramolecular contacts, makes IDRs inherently sensitive to their chemical environment. We report a combined experimental, computational, and analytical framework for high-throughput characterization of IDR sensitivity. Our framework reveals that IDRs can expand or compact in response to changes in their solution environment. Importantly, the direction and magnitude of conformational change depend on both protein sequence and cosolute identity. For example, some solutes such as short polyethylene glycol chains exert an expanding effect on some IDRs and a compacting effect on others. Despite this complex behavior, we can rationally interpret IDR responsiveness to solution composition changes using relatively simple polymer models. Our results imply that solution-responsive IDRs are ubiquitous and can provide an additional layer of regulation to biological systems.

Controlling Structural Bias in Intrinsically Disordered Proteins Using Solution Space Scanning.

Intrinsically disordered proteins or regions (IDRs) differ from their well-folded counterparts by lacking a stable tertiary state. Instead, IDRs exist in an ensemble of conformations and often possess localized, loosely held residual structure, which can be a key determinant of their activity. With no extensive network of noncovalent bonds and a high propensity for exposed surface areas, various features of an IDR's ensemble-including the local residual structure and global conformational biases-are an emergent property of both the amino acid sequence and the solution environment. Here, we attempt to understand how shifting solution conditions can alter an IDR's ensemble. We present an efficient computational method to alter solution-protein interactions we term Solution Space (SolSpace) Scanning. SolSpace scanning uses all-atom Monte Carlo simulations to construct ensembles under a wide range of distinct solution conditions. We find that by tuning the interactions of specific protein moieties with the solution in a systematic manner, we can not only alter IDR global dimensions but also completely change the local residual structure in a sequence. SolSpace scanning therefore offers an alternative approach to mutational studies for exploring sequence-to-ensemble relationships in IDRs. Our results raise the possibility of solution-based regulation of IDR functions both outside and within the dynamic environment of cells.

Phase separation of YAP reorganizes genome topology for long-term YAP target gene expression.

Yes-associated protein (YAP) is a transcriptional co-activator that regulates cell proliferation and survival by binding to a select set of enhancers for target gene activation. How YAP coordinates these transcriptional responses is unknown. Here, we demonstrate that YAP forms liquid-like condensates in the nucleus. Formed within seconds of hyperosmotic stress, YAP condensates compartmentalized the YAP transcription factor TEAD1 and other YAP-related co-activators, including TAZ, and subsequently induced the transcription of YAP-specific proliferation genes. Super-resolution imaging using assay for transposase-accessible chromatin with photoactivated localization microscopy revealed that the YAP nuclear condensates were areas enriched in accessible chromatin domains organized as super-enhancers. Initially devoid of RNA polymerase II, the accessible chromatin domains later acquired RNA polymerase II, transcribing RNA. The removal of the intrinsically-disordered YAP transcription activation domain prevented the formation of YAP condensates and diminished downstream YAP signalling. Thus, dynamic changes in genome organization and gene activation during YAP reprogramming is mediated by liquid-liquid phase separation.

Cell Volume Controls Protein Stability and Compactness of the Unfolded State.

Macromolecular crowding is widely accepted as one of the factors that can alter protein stability, structure, and function inside cells. Less often considered is that crowding can be dynamic: as cell volume changes, either as a result of external duress or in the course of the cell cycle, water moves in or out through membrane channels, and crowding changes in tune. Both theory and in vitro experiments predict that protein stability will be altered as a result of crowding changes. However, it is unclear how much the structural ensemble is altered as crowding changes in the cell. To test this, we look at the response of a FRET-labeled kinase to osmotically induced volume changes in live cells. We examine both the folded and unfolded states of the kinase by changing the temperature of the media surrounding the cell. Our data reveals that crowding compacts the structure of its unfolded ensemble but stabilizes the folded protein. We propose that the structure of proteins lacking a rigid, well-defined tertiary structure could be highly sensitive to both increases and decreases in cell volume. Our findings present a possible mechanism for disordered proteins to act as sensors and actuators of cell cycle or external stress events that coincide with a change in macromolecular crowding.