Charged sequence motifs increase the propensity towards liquid–liquid phase separation

Szabó, András László; Sánta, Anna; Pancsa, Rita; Gáspári, Zoltán

doi:10.1002/1873-3468.14294

Cited by 8 publications

(14 citation statements)

References 53 publications

(75 reference statements)

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“…At temperatures below the approximated phase boundary indicated by the temperature transitions ( Figure 6C , Figure 7—figure supplement 1 ), some p-FRQ phase separation had already occurred in the phase-separation buffer. Thus, FRQ phosphorylation alters its behavior consistent with LLPS as evidenced by its non-zero, concentration-dependent absorbance at lower temperatures, most likely by increasing the relative negative charges, changing the charge patterns, and providing more sites for multivalent interactions within the molecule ( Figure 6C , Figure 6—figure supplement 5 , Figure 7—figure supplement 1 ; Somjee et al, 2020 ; Szabó et al, 2022 ).…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

Tariq,

Maurici,

Bartholomai

et al. 2024

eLife

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Circadian clocks are composed from molecular oscillators that pace rhythms of gene expression to the diurnal cycle. Therein, transcriptional-translational negative feedback loops (TTFLs) generate oscillating levels of transcriptional repressor proteins that regulate their own gene expression. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH) and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the White-Collar complex (WCC). A key question concerns how FRQ orchestrates molecular interactions at the core of the clock despite containing little predicted tertiary structure. We present the reconstitution and biophysical characterization of FRQ and the FFC in unphosphorylated and highly phosphorylated states. Site-specific spin labeling and pulse-dipolar ESR spectroscopy provides domain-specific structural details on the full-length, 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino-acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic circadian clocks.

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Section: Resultsmentioning

confidence: 99%

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

Tariq,

Maurici,

Bartholomai

et al. 2024

eLife

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show abstract

“…Phosphorylation of FRQ substantially enhanced its ability to phase separate, mostly likely by increasing the relative negative charges, altering the charge patterns, and providing more sites for multivalent interactions within the molecule ( SI Appendix , Fig. S17)(51, 54).…”

Section: Resultsmentioning

confidence: 99%

“…These two properties, although related, are not directly correlated (49, 50, 53). In particular, a preponderance of aromatic and non-aromatic π-π planar contacts and charge-dense or charge-repeat regions drive LLPS (50, 53) and these features are consistently found in clock repressors, particularly FRQ. The similar amino acid compositions of these proteins may then potentially give rise to their shared function.…”

Section: Discussionmentioning

confidence: 99%

“…The similar amino acid compositions of these proteins may then potentially give rise to their shared function. Moreover, extensive phosphorylation may alter LLPS propensity because it increases extended states, solvation of the peptide backbone, charge density and intermolecular hydrogen bonding capacity (49, 50, 53). For FRQ, the sequence predictions are borne out experimentally, with p-FRQ displaying a different phase behavior relative to np-FRQ.…”

Section: Discussionmentioning

confidence: 99%

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Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

Tariq

Maurici

Bartholomai

et al. 2022

Preprint

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Circadian clocks are composed from molecular oscillators that pace rhythms of gene expression to the diurnal cycle. Therein, transcriptional-translational negative feedback loops (TTFLs) generate oscillating levels of transcriptional repressor proteins that regulate their own gene expression. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH) and Casein-Kinase I (CK1) form the FFC complex that acts to repress expression of clock-controlled genes activated by the White-Collar complex (WCC). A key question concerns how the FRQ protein orchestrates molecular interactions at the core of the clock despite containing little tertiary structure. We present the reconstitution and biophysical characterization of FRQ and the FFC complex in unphosphorylated and highly phosphorylated states. An integrated structural and computational biology approach incorporating site-specific spin labeling and pulse-dipolar ESR spectroscopy provides domain-specific structural details on FRQ and the FFC. FRQ, although substantially disordered, contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. Phosphorylation of FRQ increases conformational flexibility and alters oligomeric state but the resulting changes in structure and dynamics are non-uniform. FRQ undergoes liquid-liquid phase separation (LLPS) in a phosphorylation dependent manner to sequester FRH and CK1 and influence CK1 enzymatic activity. Live imaging of Neurospora hyphae reveals FRQ foci near the nuclear periphery characteristic of condensates. Analog clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino-acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic circadian clocks.

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“…Low-complexity regions (LCRs) have in the past decade experienced a surge of attention, driven by the observation that they are associated with mesoscale organization in cells: clusters, granules, hydrogels [15][16][17], membraneless organelles, and a host of related structures now referred to as biomolecular condensates [18]. Charged LCRs in particular play a crucial role in biomolecular condensation in highly influential model systems, mediating complex coacervation [7] and phase separation [19][20][21][22]. Particular sequence features such as enrichment with positively charged residues like arginine and with conformationally flexible glycine, most memorably in the RGG motif [23], appear often in RNA binding proteins that are known to condense; interactions with cationic residues can be modulated by negatively charged regions, leading to the proposal of a molecular grammar for such interactions [24].…”

Section: Introductionmentioning

confidence: 99%

Pervasive, conserved secondary structure in highly charged protein regions

Triandafillou

Pan

Dinner

et al. 2023

Preprint

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Understanding how protein sequences confer function remains a defining challenge in molecular biology. Two approaches have yielded enormous insight yet are often pursued separately: structure-based, where sequence-encoded structures mediate function, and disorder-based, where sequences dictate physicochemical and dynamical properties which determine function in the absence of stable structure. Here we study highly charged protein regions (>40% charged residues), which are routinely presumed to be disordered. Using recent advances in structure prediction and experimental structures, we show that roughly 40% of these regions form well-structured helices. Features often used to predict disorder—high charge density, low hydrophobicity, low sequence complexity, and evolutionarily varying length—are also compatible with solvated, variable-length helices. We show that a simple composition classifier predicts the existence of structure far better than well-established heuristics based on charge and hydropathy. We show that helical structure is more prevalent than previously appreciated in highly charged regions of diverse proteomes and characterize the conservation of highly charged regions. Our results underscore the importance of integrating, rather than choosing between, structure- and disorder-based approaches.

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Charged sequence motifs increase the propensity towards liquid–liquid phase separation

Cited by 8 publications

References 53 publications

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

Pervasive, conserved secondary structure in highly charged protein regions

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