Amyloid-Forming Segment Induces Aggregation of FUS-LC Domain from Phase Separation Modulated by Site-Specific Phosphorylation

The RNA-binding protein fused in sarcoma (FUS) forms physiological granules and pathological fibrils, which facilitate RNA functions and cause neurodegenerative diseases, respectively. Phosphorylation at Ser/Thr residues may regulate the functional assembly of FUS and prevent pathological aggregation in cells. However, the low-complexity nature of the FUS sequence makes it challenging to characterize how phosphorylation of specific sites within the core amyloid-forming segment affects aggregation. Taking advantage of the recently solved molecular structures of the fibrillar core of the FUS low-complexity (FUS-LC) domain, we systematically investigated the aggregation of repeated segments within the core. We identified a segment with a strong amyloid-forming tendency that induced the aggregation of FUS-LC domain in phase-separated liquid droplets and further seeded the aggregation of full-length FUS. The aggregation propensity and seeding ability of this amyloid-forming segment were modulated by site-specific phosphorylation. Solid-state nuclear magnetic resonance (NMR) spectroscopy and computational modeling implied that site-specific phosphorylation at Ser61 plays key roles in FUS assembly by disrupting both intra- and intermolecular interactions that maintain the amyloid core structure. [Display omitted] •A segment with strong tendency to form amyloid fibrils of FUS was explored.•The segment can convert phase separated droplets to pathological fibrils.•Phosphorylation at Ser61 could inhibit the aggregation of the segment.•Phos-Ser61 could prohibit the seeding effects due to its retarded fibril formation process.•Phos-Ser61 disrupts both intra- and intermolecular interactions of the segment.