scholarly journals Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation

2020 ◽  
Author(s):  
Erik W. Martin ◽  
F. Emil Thomasen ◽  
Nicole M. Milkovic ◽  
Matthew J. Cuneo ◽  
Christy R. Grace ◽  
...  
Keyword(s):  
Phase Separation ◽  
Phase Behavior ◽  
Rna Binding ◽  
Mechanistic Explanation ◽  
Md Simulations ◽  
Low Complexity ◽  
Coarse Grained ◽  
Intrinsically Disordered ◽  
X Ray Scattering

AbstractLiquid-liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (1) electrostatically mediated interactions that compact hnRNPA1 and contribute to phase separation, and (2) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation.Graphical abstracthnRNPA1 phase separation is highly salt sensitive.Phase separation of the low-complexity domain (LCD) of hnRNPA1 increases with NaCl. In contrast, phase separation of full-length hnRNPA1 is saltsensitive. At low NaCl concentrations, electrostatic RRM-LCD interactions occur and can contribute positively to phase separation, but they are screened at high NaCl concentrations. The folded domains solubilize hnRNPA1 under these conditions and prevent phase separation.

scholarly journals Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation

2021 ◽  
Author(s):  
Erik W Martin ◽  
F Emil Thomasen ◽  
Nicole M Milkovic ◽  
Matthew J Cuneo ◽  
Christy R Grace ◽  
...  
Keyword(s):  
Phase Separation ◽  
Phase Behavior ◽  
Rna Binding ◽  
Mechanistic Explanation ◽  
Md Simulations ◽  
Low Complexity ◽  
Coarse Grained ◽  
Intrinsically Disordered ◽  
X Ray Scattering

Abstract Liquid–liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (i) electrostatically-mediated interactions that compact hnRNPA1 and contribute to phase separation and (ii) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation.

scholarly journals Deciphering how naturally occurring sequence features impact the phase behaviors of disordered prion-like domains

2021 ◽  
Author(s):  
Anne Bremer ◽  
Mina Farag ◽  
Wade M. Borcherds ◽  
Ivan Peran ◽  
Erik W. Martin ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Driving Forces ◽  
Rna Binding Proteins ◽  
Low Complexity ◽  
Coarse Grained ◽  
Coarse Grained Model ◽  
Intrinsically Disordered ◽  
Lysine Residues ◽  
Arginine Residues

AbstractPhase separation of intrinsically disordered prion-like low-complexity domains (PLCDs) derived from RNA-binding proteins enable the formation of biomolecular condensates in cells. PLCDs have distinct amino acid compositions, and here we decipher the physicochemical impact of conserved compositional biases on the driving forces for phase separation. We find that tyrosine residues make for stronger drivers of phase separation than phenylalanine. Depending on their sequence contexts, arginine residues enhance or weaken phase separation, whereas lysine residues weaken cohesive interactions within PLCDs. Increased net charge per residue (NCPR) weakens the driving forces for phase separation of PLCDs and this effect can be modeled quantitatively. The effects of NCPR also weaken known correlations between the dimensions of single chains in dilute solution and the driving forces for phase separation. We build on experimental data to develop a coarse-grained model for accurate simulations of phase separation that yield novel insights regarding PLCD phase behavior.

scholarly journals Molecular details of protein condensates probed by microsecond-long atomistic simulations

2020 ◽  
Author(s):  
Wenwei Zheng ◽  
Gregory L. Dignon ◽  
Xichen Xu ◽  
Roshan M. Regy ◽  
Nicolas L. Fawzi ◽  
...  
Keyword(s):  
Phase Separation ◽  
Intrinsically Disordered Proteins ◽  
Low Complexity ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Residue Interaction ◽  
Coarse Grained Simulations ◽  
Protein Density

AbstractThe formation of membraneless organelles in cells commonly occurs via liquid-liquid phase separation (LLPS), and is in many cases driven by multivalent interactions between intrinsically disordered proteins (IDPs). Molecular simulations can reveal the specific amino acid interactions driving LLPS, which is hard to obtain from experiment. Coarse-grained simulations have been used to directly observe the sequence determinants of phase separation but have limited spatial resolution, while all-atom simulations have yet to be applied to LLPS due to the challenges of large system sizes and long time scales relevant to phase separation. We present a novel multiscale computational framework by obtaining initial molecular configurations of a condensed protein-rich phase from equilibrium coarse-grained simulations, and back mapping to an all-atom representation. Using the specialized Anton 2 supercomputer, we resolve microscopic structural and dynamical details of protein condensates through microsecond-scale all-atom explicit-solvent simulations. We have studied two IDPs which phase separate in vitro: the low complexity domain of FUS and the N-terminal disordered domain of LAF-1. Using this approach, we explain the partitioning of ions between phases with low and high protein density, demonstrate that the proteins are remarkably dynamic within the condensed phase, identify the key residue-residue interaction modes stabilizing the dense phase, all while showing good agreement with experimental observations. Our approach is generally applicable to all-atom studies of other single and multi-component systems of proteins and nucleic acids involved in the formation of membraneless organelles.

scholarly journals Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

2020 ◽  
Vol 117 (21) ◽  
pp. 11421-11431 ◽  
Author(s):  
Benjamin S. Schuster ◽  
Gregory L. Dignon ◽  
Wai Shing Tang ◽  
Fleurie M. Kelley ◽  
Aishwarya Kanchi Ranganath ◽  
...  
Keyword(s):  
Phase Separation ◽  
Intrinsically Disordered Proteins ◽  
Lipid Membrane ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Sequence Features ◽  
Intrinsically Disordered ◽  
Arginine Residues

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

scholarly journals A predictive coarse-grained model for position-specific effects of post-translational modifications on disordered protein phase separation

2020 ◽  
Author(s):  
T. M. Perdikari ◽  
N. Jovic ◽  
G. L. Dignon ◽  
Y. C. Kim ◽  
N. L. Fawzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Phase Separation ◽  
Phase Behavior ◽  
Intrinsically Disordered Proteins ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Post Translational Modifications ◽  
Coarse Grained Model ◽  
Intrinsically Disordered ◽  
Liquid Liquid Phase Separation

AbstractBiomolecules undergo liquid-liquid phase separation (LLPS) resulting in the formation of multicomponent protein-RNA membraneless organelles in cells. However, the physiological and pathological role of post translational modifications (PTMs) on the biophysics of phase behavior is only beginning to be probed. To study the effect of PTMs on LLPS in silico, we extend our transferable coarse-grained model of intrinsically disordered proteins to include phosphorylated and acetylated amino acids. Using the parameters for modified amino acids available for fixed charge atomistic forcefields, we parameterize the size and atomistic hydropathy of the coarse-grained modified amino acid beads, and hence the interactions between the modified and natural amino acids. We then elucidate how the number and position of phosphorylated and acetylated residues alter the protein’s single chain compactness and its propensity to phase separate. We show that both the number and the position of phosphorylated threonines/serines or acetylated lysines can serve as a molecular on/off switch for phase separation in the well-studied disordered regions of FUS and DDX3X, respectively. We also compare modified residues to their commonly used PTM mimics for their impact on chain properties. Importantly, we show that the model can predict and capture experimentally measured differences in the phase behavior for position-specific modifications, showing that the position of modifications can dictate phase separation. In sum, this model will be useful for studying LLPS of post-translationally modified intrinsically disordered proteins and predicting how modifications control phase behavior with position-specific resolution.Statement of SignificancePost-translational modifications are important regulators of liquid-liquid phase separation (LLPS) which drives the formation of biomolecular condensates. Theoretical methods can be used to characterize the biophysical properties of intrinsically disordered proteins (IDPs). Our recent framework for molecular simulations using a Cα-centered coarse-grained model can predict the effect of various perturbations such as mutations (Dignon et al. PloS Comput. Biol, 2018) and temperature (Dignon et al, ACS Cent. Sci., 2019) on LLPS. Here, we expand this framework to incorporate modified residues like phosphothreonine, phosphoserine and acetylysine. This model will prove useful for simulating the phase separation of post-translationally modified IDPs and predicting how position-specific modifications can control phase behavior across the large family of proteins known to be phosphorylated and acetylated.

scholarly journals Sequence dependent co-phase separation of RNA-protein mixtures elucidated using molecular simulations

2020 ◽  
Author(s):  
Roshan Mammen Regy ◽  
Gregory L. Dignon ◽  
Wenwei Zheng ◽  
Young Chan Kim ◽  
Jeetain Mittal
Keyword(s):  
Phase Separation ◽  
Intrinsically Disordered Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Experimental Studies ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Sequence Specificity ◽  
Intrinsically Disordered ◽  
Modelling Framework

ABSTRACTRibonucleoprotein (RNP) granules are membraneless organelles (MLOs) which majorly consist of RNA and RNA-binding proteins and are formed via liquid-liquid phase separation (LLPS). Experimental studies investigating the drivers of LLPS have shown that intrinsically disordered proteins (IDPs) and nucleic acids like RNA play a key role in modulating protein phase separation. There is currently a dearth of modelling techniques which allow one to delve deeper into how RNA plays its role as a modulator/promoter of LLPS in cells using computational methods. Here we present a coarse-grained RNA model developed to fill this gap, which together with our recently developed HPS model for protein LLPS, allows us to capture the factors driving RNA-protein co-phase separation. We explore the capabilities of the modelling framework with the LAF-1 RGG/RNA system which has been well studied in experiments and also with the HPS model previously. Further taking advantage of the fact that the HPS model maintains sequence specificity we explore the role of charge patterning on controlling RNA incorporation into condensates. With increased charge patterning we observe formation of structured or patterned condensates which suggests the possible roles of RNA in not only shifting the phase boundaries but also introducing microscopic organization in MLOs.

scholarly journals Model for disordered proteins with strongly sequence-dependent liquid phase behavior

2019 ◽  
Author(s):  
Antonia Statt ◽  
Helena Casademunt ◽  
Clifford P. Brangwynne ◽  
Athanassios Z. Panagiotopoulos
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Phase Behavior ◽  
Intrinsically Disordered Proteins ◽  
Protein A ◽  
Liquid Phase Separation ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Liquid Liquid Phase Separation

Phase separation of intrinsically disordered proteins is important for the formation of membraneless organelles, or biomolecular condensates, which play key roles in the regulation of biochemical processes within cells. In this work, we investigated the phase separation of different sequences of a coarse-grained model for intrinsically disordered proteins and discovered a surprisingly rich phase behavior. We studied both the fraction of total hydrophobic parts and the distribution of hydrophobic parts. Not surprisingly, sequences with larger hydrophobic fractions showed conventional liquid-liquid phase separation. The location of the critical point was systematically influenced by the terminal beads of the sequence, due to changes in interfacial composition and tension. For sequences with lower hydrophobicity, we observed not only conventional liquid-liquid phase separation, but also reentrant phase behavior, in which the liquid phase density decreases at lower temperatures. For some sequences, we observed formation of open phases consisting of aggregates, rather than a normal liquid. These aggregates had overall lower densities than the conventional liquid phases, and exhibited complex geometries with large interconnected string-like or membrane-like clusters. Our findings suggest that minor alterations in the ordering of residues may lead to large changes in the phase behavior of the protein, a fact of significant potential relevance for biology.

scholarly journals Low-complexity domain of U1-70K modulates phase separation and aggregation through distinctive basic-acidic motifs

2019 ◽  
Vol 5 (11) ◽  
pp. eaax5349 ◽  
Author(s):  
Song Xue ◽  
Rui Gong ◽  
Fanqi He ◽  
Yanqin Li ◽  
Yunjia Wang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Low Complexity ◽  
Protein Domain ◽  
Shed Light ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation (LLPS) facilitates the formation of functional membraneless organelles and recent reports have linked this phenomenon to protein aggregation in neurodegenerative diseases. Understanding the mechanism of LLPS and its regulation thus promises to shed light on the pathogenesis of these conditions. The RNA-binding protein U1-70K, which aggregates in brains of Alzheimer’s disease patients, is considered a potential target for Alzheimer’s therapy. Here, we report that two fragments in the low-complexity (LC) domain of U1-70K can undergo LLPS. We have demonstrated that the repetitive basic-acidic motifs in these fragments induce nucleotide-independent phase separation and initiate aggregation in vitro. We also have confirmed that LLPS and aggregation occur in vivo and that the content of ampholytic motifs in a protein domain determines the transition between droplets and aggregation, providing insights into the mechanism underlying the formation of diverse assembly states.

scholarly journals Relation between single-molecule properties and phase behavior of intrinsically disordered proteins

2018 ◽  
Vol 115 (40) ◽  
pp. 9929-9934 ◽  
Author(s):  
Gregory L. Dignon ◽  
Wenwei Zheng ◽  
Robert B. Best ◽  
Young C. Kim ◽  
Jeetain Mittal
Keyword(s):  
Phase Separation ◽  
Phase Behavior ◽  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Critical Role ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Phase Boundaries ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions

Proteins that undergo liquid–liquid phase separation (LLPS) have been shown to play a critical role in many physiological functions through formation of condensed liquid-like assemblies that function as membraneless organelles within biological systems. To understand how different proteins may contribute differently to these assemblies and their functions, it is important to understand the molecular driving forces of phase separation and characterize their phase boundaries and material properties. Experimental studies have shown that intrinsically disordered regions of these proteins are a major driving force, as many of them undergo LLPS in isolation. Previous work on polymer solution phase behavior suggests a potential correspondence between intramolecular and intermolecular interactions that can be leveraged to discover relationships between single-molecule properties and phase boundaries. Here, we take advantage of a recently developed coarse-grained framework to calculate the θ temperatureTθ, the Boyle temperatureTB, and the critical temperatureTcfor 20 diverse protein sequences, and we show that these three properties are highly correlated. We also highlight that these correlations are not specific to our model or simulation methodology by comparing between different pairwise potentials and with data from other work. We, therefore, suggest that smaller simulations or experiments to determineTθorTBcan provide useful insights into the corresponding phase behavior.

scholarly journals Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Jarrett Smith ◽  
Deepika Calidas ◽  
Helen Schmidt ◽  
Tu Lu ◽  
Dominique Rasoloson ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Intrinsically Disordered Protein ◽  
General Mechanism ◽  
P Granules ◽  
Rna Granules ◽  
Intrinsically Disordered ◽  
Disordered Protein

RNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings suggest that MEX-5 interferes with MEG-3’s access to RNA, thus locally suppressing MEG-3 phase separation to drive P granule asymmetry. Regulated access to RNA, combined with RNA-induced phase separation of key scaffolding proteins, may be a general mechanism for controlling the formation of RNA granules in space and time.

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