Intracellular environments are heterogeneous milieus comprised of macromolecules, osmolytes, and a range of assemblies that include membrane-bound organelles and membraneless biomolecular ...condensates. The latter are nonstoichiometric assemblies of protein and RNA molecules. They represent distinct phases and form via intracellular phase transitions. Here, we present insights from recent studies and provide a perspective on how phase transitions that lead to biomolecular condensates might contribute to cellular functions.
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IJS, KILJ, NUK, PNG, UL, UM
Prion-like domains (PLDs) can drive liquid-liquid phase separation (LLPS) in cells. Using an integrative biophysical approach that includes nuclear magnetic resonance spectroscopy, small-angle x-ray ...scattering, and multiscale simulations, we have uncovered sequence features that determine the overall phase behavior of PLDs. We show that the numbers (valence) of aromatic residues in PLDs determine the extent of temperature-dependent compaction of individual molecules in dilute solutions. The valence of aromatic residues also determines full binodals that quantify concentrations of PLDs within coexisting dilute and dense phases as a function of temperature. We also show that uniform patterning of aromatic residues is a sequence feature that promotes LLPS while inhibiting aggregation. Our findings lead to the development of a numerical stickers-and-spacers model that enables predictions of full binodals of PLDs from their sequences.
Liquid-liquid phase separation, driven by collective interactions among multivalent and intrinsically disordered proteins, is thought to mediate the formation of membrane-less organelles in cells. ...Using parallel cellular and in vitro assays, we show that the Nephrin intracellular domain (NICD), a disordered protein, drives intracellular phase separation via complex coacervation, whereby the negatively charged NICD co-assembles with positively charged partners to form protein-rich dense liquid droplets. Mutagenesis reveals that the driving force for phase separation depends on the overall amino acid composition and not the precise sequence of NICD. Instead, phase separation is promoted by one or more regions of high negative charge density and aromatic/hydrophobic residues that are distributed across the protein. Many disordered proteins share similar sequence characteristics with NICD, suggesting that complex coacervation may be a widely used mechanism to promote intracellular phase separation.
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•Disordered Nephrin intracellular domain (NICD) forms phase-separated nuclear bodies•NICD phase separates via complex coacervation•Aromatic/hydrophobic residues and high (−) charge density promote phase separation•Disordered regions with NICD-like sequence features are common in human proteome
Pak et al. describe cellular liquid-liquid phase separation of a negatively charged intrinsically disordered protein, the Nephrin intracellular domain. Phase separation is driven by co-assembly with positively charged partners, a process termed complex coacervation. Disordered regions with NICD-like sequence features are common in the human proteome, suggesting complex coacervation may be widespread.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino ...acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences.
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•Phase separation of FUS requires both the N-terminal PLD and the C-terminal RBD•Tyrosine and arginine govern the saturation concentration of phase separation•Glycine maintains liquidity, whereas glutamine and serine promote hardening•An associative polymer model predicts the phase behavior of FUS family proteins
The phase-separation behavior of FUS family proteins can be predicted by the prevalence and position of specific amino acids.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Phase transitions of linear multivalent proteins control the reversible formation of many intracellular membraneless bodies. Specific non-covalent crosslinks involving domains/motifs lead to ...system-spanning networks referred to as gels. Gelation transitions can occur with or without phase separation. In gelation driven by phase separation multivalent proteins and their ligands condense into dense droplets, and gels form within droplets. System spanning networks can also form without a condensation or demixing of proteins into droplets. Gelation driven by phase separation requires lower protein concentrations, and seems to be the biologically preferred mechanism for forming membraneless bodies. Here, we use coarse-grained computer simulations and the theory of associative polymers to uncover the physical properties of intrinsically disordered linkers that determine the extent to which gelation of linear multivalent proteins is driven by phase separation. Our findings are relevant for understanding how sequence-encoded information in disordered linkers influences phase transitions of multivalent proteins.
There is growing interest in the topic of intracellular phase transitions that lead to the formation of biologically regulated biomolecular condensates. These condensates are membraneless bodies ...formed by phase separation of key protein and nucleic acid molecules from the cytoplasmic or nucleoplasmic milieus. The drivers of phase separation are referred to as scaffolds whereas molecules that preferentially partition into condensates formed by scaffolds are known as clients. Recent advances have shown that it is possible to generate physical and functional facsimiles of many biomolecular condensates in vitro. This is achieved by titrating the concentration of key scaffold proteins and solution parameters such as salt concentration, pH, or temperature. The ability to reproduce phase separation in vitro allows one to compare the relationships between information encoded in the sequences of scaffold proteins and the driving forces for phase separation. Many scaffold proteins include intrinsically disordered regions whereas others are entirely disordered. Our focus is on comparative assessments of phase separation for different scaffold proteins, specifically intrinsically disordered linear multivalent proteins. We highlight the importance of coexistence curves known as binodals for quantifying phase behavior and comparing driving forces for sequence-specific phase separation. We describe the information accessible from full binodals and highlight different methods for-and challenges associated with-mapping binodals. In essence, we provide a wish list for in vitro characterization of phase separation of intrinsically disordered proteins. Fulfillment of this wish list through key advances in experiment, computation, and theory should bring us closer to being able to predict in vitro phase behavior for scaffold proteins and connect this to the functions and features of biomolecular condensates.
The transcriptional co-activator p300 is a histone acetyltransferase (HAT) that is typically recruited to transcriptional enhancers and regulates gene expression by acetylating chromatin. Here we ...show that the activation of p300 directly depends on the activation and oligomerization status of transcription factor ligands. Using two model transcription factors, IRF3 and STAT1, we demonstrate that transcription factor dimerization enables the trans-autoacetylation of p300 in a highly conserved and intrinsically disordered autoinhibitory lysine-rich loop, resulting in p300 activation. We describe a crystal structure of p300 in which the autoinhibitory loop invades the active site of a neighbouring HAT domain, revealing a snapshot of a trans-autoacetylation reaction intermediate. Substrate access to the active site involves the rearrangement of an autoinhibitory RING domain. Our data explain how cellular signalling and the activation and dimerization of transcription factors control the activation of p300, and therefore explain why gene transcription is associated with chromatin acetylation.
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KISLJ, NUK, SBMB, UL, UM, UPUK
Despite the important role of prion domains in neurodegenerative disease, their physiological function has remained enigmatic. Previous work with yeast prions has defined prion domains as sequences ...that form self-propagating aggregates. Here, we uncovered an unexpected function of the canonical yeast prion protein Sup35. In stressed conditions, Sup35 formed protective gels via pH-regulated liquid-like phase separation followed by gelation. Phase separation was mediated by the N-terminal prion domain and regulated by the adjacent pH sensor domain. Phase separation promoted yeast cell survival by rescuing the essential Sup35 translation factor from stress-induced damage. Thus, prion-like domains represent conserved environmental stress sensors that facilitate rapid adaptation in unstable environments by modifying protein phase behavior.
The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we ...combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.
Intracellular biomolecular condensates are membraneless organelles that encompass large numbers of multivalent protein and nucleic acid molecules. The bodies assemble via a combination of ...liquid-liquid phase separation and gelation. A majority of condensates included multiple components and show multilayered organization as opposed to being well-mixed unitary liquids. Here, we put forward a simple thermodynamic framework to describe the emergence of spatially organized droplets in multicomponent systems comprising of linear multivalent polymers also known as associative polymers. These polymers, which mimic proteins and/or RNA have the architecture of domains or motifs known as stickers that are interspersed by flexible spacers known as linkers. Using a minimalist numerical model for a four-component system, we have identified features of linear multivalent molecules that are necessary and sufficient for generating spatially organized droplets. We show that differences in sequence-specific effective solvation volumes of disordered linkers between interaction domains enable the formation of spatially organized droplets. Molecules with linkers that are preferentially solvated are driven to the interface with the bulk solvent, whereas molecules that have linkers with negligible effective solvation volumes form cores in the core-shell architectures that emerge in the minimalist four-component systems. Our modeling has relevance for understanding the physical determinants of spatially organized membraneless organelles.
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