Quantitative description of the phase-separation behavior of the multivalent SLP65–CIN85 complex
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PROVIDER: S-EPMC10923291 | biostudies-literature | 2024 Feb
REPOSITORIES: biostudies-literature
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Project description:Multivalent binding is essential to many biological processes because it builds high-affinity bonds by using several weak binding interactions simultaneously. Multivalent polymers have shown promise as inhibitors of toxins and other pathogens, and they are important components in the formation of biocondensates. Explaining how structural features of these polymers change their binding and subsequent control of phase separation is critical to designing better pathogen inhibitors and also to understanding diseases associated with membraneless organelles. In this work, we will examine the binding of a multivalent polymer to a small target. This scenario could represent a polymeric inhibitor binding to a toxic protein or RNA binding to an RNA-binding protein in the case of liquid-liquid phase separation. We use simulation and theory to show that flexible random-coil polymers bind more strongly than stiff rod-like polymers and that flexible polymers nucleate condensed phases at lower binding energies than their rigid analogs. We hope these results will provide insight into the rational design of polymeric inhibitors and improve our understanding of phase separation in cells and membraneless organelles.
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Project description:Multivalent polymers are a key structural component of many biocondensates. When interacting with their cognate binding proteins, multivalent polymers such as RNA and modular proteins have been shown to influence the liquid-liquid phase separation (LLPS) boundary to both control condensate formation and to influence condensate dynamics after phase separation. Much is still unknown about the function and formation of these condensed droplets, but changes in their dynamics or phase separation are associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's Disease. Therefore, investigation into how the structure of multivalent polymers relates to changes in biocondensate formation and maturation is essential to understanding and treating these diseases. Here, we use a coarse-grain, Brownian Dynamics simulation with reactive binding that mimics specific interactions in order to investigate the difference between non-specific and specific multivalent binding polymers. We show that non-specific binding interactions can lead to much larger changes in droplet formation at lower protein-polymer interaction energies than their specific, valence-limited counterparts. We also demonstrate the effects of solvent conditions and polymer length on phase separation, and we present how modulating binding energy to the polymer can change the organization of a droplet in a three component system of polymer, binding protein, and solvent. Finally, we compare the effects of surface tension and polymer binding on the condensed phase dynamics, and show that both lower protein solubilities and higher attraction/affinity of the protein to the polymer result in slower droplet dynamics. This research will help to better understand experimental systems and provides additional insight into how multivalent polymers can control LLPS.
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Project description:The thiourea-iodate reaction has been investigated simultaneously by ultraviolet-visible spectroscopy and high-performance liquid chromatography (HPLC). Absorbance-time traces measured at the isosbestic point of the iodine-triiodide system have revealed a special dual-clock behavior. During the first kinetic stage of the title reaction, iodine suddenly appears only after a well-defined time lag when thiourea is totally consumed due to the rapid thiourea-iodine system giving rise to a substrate-depletive clock reaction. After this delay, iodine in the system starts to build up suddenly to a certain level, where the system remains for quite a while. During this period, hydrolysis of formamidine disulfide as well as the formamidine disulfide-iodine system along with the Dushman reaction and subsequent reactions of the intermediates governs the parallel formation and disappearance of iodine, resulting in a fairly constant absorbance. The kinetic phase mentioned above is then followed by a more slowly increasing sigmoidally shaped profile that is characteristic of autocatalysis-driven clock reactions. HPLC studies have clearly shown that the thiourea dioxide-iodate system is responsible mainly for the latter characteristics. Of course, depending on the initial concentration ratio of the reactants, the absorbance-time curve may level off or reach a maximum followed by a declining phase. With an excess of thiourea, iodine may completely disappear from the solution as a result of the thiourea dioxide-iodine reaction. In the opposite case, with an excess of iodate, the final absorbance reaches a finite value, and at the same time, iodide ion will disappear completely from the solution due to the well-known Dushman (iodide-iodate) reaction. In addition, we have also shown that in the case of the formamidine disulfide-iodine reaction, unexpectedly the triiodide ion is more reactive toward formamidine disulfide than iodine. This feature can readily be interpreted by the enhancement of the rate of formation of the transition complex containing oppositely charged reactants. A 25-step kinetic model is proposed with just 10 fitted parameters to fit the 68 kinetic traces measured in the thiourea-iodate system and the second, but slower, kinetic phase of the thiourea-iodine reaction. The comprehensive kinetic model is constituted in such a way as to remain coherent in quantitatively describing all of the most important characteristics of the formamidine disulfide-iodine, thiourea dioxide-iodine, and thiourea dioxide-iodate systems.
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Project description:TDP-43 is an RNA-binding protein that forms ribonucleoprotein condensates via liquid-liquid phase separation (LLPS) and regulates gene expression through specific RNA interactions. Loss of TDP-43 protein homeostasis and dysfunction are tied to neurodegenerative disorders, mainly amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Alterations of TDP-43 LLPS properties may be linked to protein aggregation. However, the mechanisms regulating TDP-43 LLPS are ill-defined, particularly how TDP-43 association with specific RNA targets regulates TDP-43 condensation remains unclear. We show that RNA binding strongly promotes TDP-43 LLPS through sequence-specific interactions. RNA-driven condensation increases with the number of adjacent TDP-43-binding sites and is also mediated by multivalent interactions involving the amino and carboxy-terminal TDP-43 domains. The physiological relevance of RNA-driven TDP-43 condensation is supported by similar observations in mammalian cellular lysate. Importantly, we find that TDP-43-RNA association maintains liquid-like properties of the condensates, which are disrupted in the presence of ALS-linked TDP-43 mutations. Altogether, RNA binding plays a central role in modulating TDP-43 condensation while maintaining protein solubility, and defects in this RNA-mediated activity may underpin TDP-43-associated pathogenesis.
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Project description:Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.
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