Ligand Effects on Phase Separation of Multivalent Macromolecules

Ruff, Kiersten M.; Dar, Furqan; Pappu, Rohit V.

doi:10.1101/2020.08.15.252346

Cited by 43 publications

(72 citation statements)

References 68 publications

(143 reference statements)

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“…S3 and S4). These results are consistent with previous literature reports 26,45 , and indicate that RRPs prefer binding to the PLP in the dense phase 51,52 , thereby enhancing PLP phase separation and impacting the condensate dynamics ( Fig. 1a-c S5a).…”

Section: Resultssupporting

confidence: 93%

“…Although PLP readily undergoes homotypic condensation in the absence of RRP, the latter did not show any sign of homotypic LLPS at the concentrations used in our study. Hence, our results that the saturation concentration of PLP decreases monotonically with [RRP] are consistent with a scenario where RRP acts as a ligand that binds to the PLP preferentially in the dense phase 51,52 . In addition to the altered phase behavior of PLP due to the presence of RRP, we observe that the PLP partition coefficient (K = C PLP Dense =C PLP Dilute ) increases with [RRP].…”

Section: Resultssupporting

confidence: 90%

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Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies

et al. 2021

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Multivalent protein-protein and protein-RNA interactions are the drivers of biological phase separation. Biomolecular condensates typically contain a dense network of multiple proteins and RNAs, and their competing molecular interactions play key roles in regulating the condensate composition and structure. Employing a ternary system comprising of a prion-like polypeptide (PLP), arginine-rich polypeptide (RRP), and RNA, we show that competition between the PLP and RNA for a single shared partner, the RRP, leads to RNA-induced demixing of PLP-RRP condensates into stable coexisting phases—homotypic PLP condensates and heterotypic RRP-RNA condensates. The morphology of these biphasic condensates (non-engulfing/ partial engulfing/ complete engulfing) is determined by the RNA-to-RRP stoichiometry and the hierarchy of intermolecular interactions, providing a glimpse of the broad range of multiphasic patterns that are accessible to these condensates. Our findings provide a minimal set of physical rules that govern the composition and spatial organization of multicomponent and multiphasic biomolecular condensates.

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Section: Resultssupporting

confidence: 93%

Section: Resultssupporting

confidence: 90%

Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies

et al. 2021

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“…These results suggest that long RNA-molecules behave like high-valency super-scaffolds which can act as a hub that networks core and non-core proteins. This behavior of long RNA chains is analogous to the condensate-stabilizing effect of multi-valent spacer-sticker ligands in a recent study by Ruff et al 53 . Similarly, the patchy particle simulation study by Espinosa et al suggests that high valency, promiscuous binders can efficiently promote phase separation 29 .…”

Section: Discussionsupporting

confidence: 61%

“…Similarly, the patchy particle simulation study by Espinosa et al suggests that high valency, promiscuous binders can efficiently promote phase separation 29 . These studies, however, model scaffolds and client molecules as collapsed structures (patchy hard spheres) 29,53 . In the proteinprotein interaction network modeled in our current work, the large valency proteins (core proteins in Table 1) cannot promote large clusters in the low endogenous concentration regime because of their specificiy.…”

Section: Discussionmentioning

confidence: 99%

Effect of RNA on morphology and dynamics of membraneless organelles

Ranganathan

Shakhnovich

2021

Preprint

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Membraneless organelles (MLOs) are spatiotemporally regulated structures that concentrate multi-valent proteins or RNA, often in response to stress. The proteins enriched within MLOs are often classified as high-valency ''scaffolds'' or low valency ''clients'', with the former being associated with a phase-separation promoting role. In this study, we employ a minimal model for P-body components, with a defined protein-protein interaction network, to study their phase-separation at biologically realistic low protein concentrations. Without RNA multivalent proteins can assemble into solid-like clusters only in the regime of high concentration and stable interactions. RNA molecules promote cluster formation in an RNA-length dependent manner, even in the regime of weak interactions and low protein volume fraction. Our simulations reveal that long RNA chains act as super-scaffolds that stabilize large RNA-protein clusters by recruiting low-valency proteins within them while also ensuring functional ``liquid-like'' turnover of components. Our results suggest that RNA-mediated phase separation could be a plausible mechanism for spatiotemporally regulated phase-separation in the cell.

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“…This is an important distinction as polyvalent ligands (e.g. long polyUb chains) may substantially alter the driving forces for UBQLN2 phase separation (Ruff et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation

Zheng

Castañeda

2021

Preprint

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Shuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N-terminal ubiquitin-like (UBL) and C-terminal ubiquitin-associated (UBA) domains, respectively. Between these two folded domains are intrinsically disordered STI1-I and STI1-II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1-II region enables UBQLN2 to undergo liquid-liquid phase separation (LLPS) to form liquid droplets in vitro and biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically-disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using NMR spectroscopy. To our surprise, aside from well-studied canonical UBL:UBA interactions, there also exist moderate and weak interactions between the UBL and STI1-I/STI1-II domains, and between the UBA domain and the linker connecting the two STI1 regions, respectively. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or STI1 domains on the phase behavior and physiological functions of UBQLN2.

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Ligand Effects on Phase Separation of Multivalent Macromolecules

Cited by 43 publications

References 68 publications

Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies

Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies

Effect of RNA on morphology and dynamics of membraneless organelles

Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation

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