Subcellular membrane-less organelles consist of proteins with low complexity domains. Many of them, such as hnRNPA1, can assemble into both a polydisperse liquid phase and an ordered solid phase of amyloid fibril. The former mirrors biological granule assembly, while the latter is usually associated with neurodegenerative disease. Here, we observe a reversible amyloid formation of hnRNPA1 that synchronizes with liquid–liquid phase separation, regulates the fluidity and mobility of the liquid-like droplets, and facilitates the recruitment of hnRNPA1 into stress granules. We identify the reversible amyloid-forming cores of hnRNPA1 (named hnRACs). The atomic structures of hnRACs reveal a distinct feature of stacking Asp residues, which contributes to fibril reversibility and explains the irreversible pathological fibril formation caused by the Asp mutations identified in familial ALS. Our work characterizes the structural diversity and heterogeneity of reversible amyloid fibrils and illuminates the biological function of reversible amyloid formation in protein phase separation.

α-Synuclein (α-Syn) can form different fibril strains with distinct polymorphs and neuropathologies, which is associated with the clinicopathological variability in synucleinopathies. How different α-syn fibril strains are produced and selected under disease conditions remains poorly understood. In this study, we show that the hereditary mutation G51D induces α-syn to form a distinct fibril strain in vitro. The cryogenic electron microscopy (cryo-EM) structure of the G51D fibril strain was determined at 2.96 Å resolution. The G51D fibril displays a relatively small and extended serpentine fold distinct from other α-syn fibril structures. Moreover, we show by cryo-EM that wild-type (WT) α-syn can assembly into the G51D fibril strain via cross-seeding with G51D fibrils. Our study reveals a distinct structure of G51D fibril strain triggered by G51D mutation but feasibly adopted by both WT and G51D α-syn, which suggests the cross-seeding and strain selection of WT and mutant α-syn in familial Parkinson’s disease (fPD).

Significance: Receptor-interacting protein kinases 3 (RIPK3), a hub player in necrotic cell death, forms amyloid-like polymers in response to upstream mediators. This work, by using cryo-EM and solid-state NMR, reports the structure of human RIPK3 amyloid fibril core. The fibril core is mainly composed of the consensus RHIM domain and exhibits distinctive properties: an exceptionally small fibril core and existence in both handedness. These traits may provide a favorable geometry for the arrangement of kinase domain on the fibril surface to boost RIPK3 phosphorylation and the following activation of downstream MLKL. This work sheds light on how functional fibrils utilize the amyloid state to spatially regulate their functional domains, which is usually not a concern of pathological fibrils.

Escherichia coli UvrD is a non‐ring‐shaped model helicase, displaying a 3′—5′ polarity in DNA unwinding. Using a transverse magnetic tweezer and DNA hairpins, we measured the unwinding kinetics of UvrD at various DNA‐destabilizing forces. The multiform patterns of unwinding bursts and the distributions of the off‐times favour the mechanism that UvrD unwinds DNA as a dimer. The two subunits of the dimer coordinate to unwind DNA processively. They can jointly switch strands and translocate backwards on the other strand to allow slow (∼40 bp/s) rewinding, or unbind simultaneously to allow quick rehybridization. Partial dissociation of the dimer results in pauses in the middle of the unwinding or increases the translocation rate from ∼40 to ∼150 nt/s in the middle of the rewinding. Moreover, the unwinding rate was surprisingly found to decrease from ∼45 to ∼10 bp/s when the force is increased from 2 to 12 pN. The results lead to a strained‐inchworm mechanism in which a conformational change that bends and tenses the ssDNA is required to activate the dimer.

Human heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) serves as a key regulating protein in RNA metabolism. Malfunction of hnRNPA1 in nucleo-cytoplasmic transport or dynamic phase separation leads to abnormal amyloid aggregation and neurodegeneration. The low complexity (LC) domain of hnRNPA1 drives both dynamic phase separation and amyloid aggregation. Here, we use cryo-electron microscopy to determine the amyloid fibril structure formed by hnRNPA1 LC domain. Remarkably, the structure reveals that the nuclear localization sequence of hnRNPA1 (termed PY-NLS), which is initially known to mediate the nucleo-cytoplamic transport of hnRNPA1 through binding with karyopherin-β2 (Kapβ2), represents the major component of the fibril core. The residues that contribute to the binding of PY-NLS with Kapβ2 also exert key molecular interactions to stabilize the fibril structure. Notably, hnRNPA1 mutations found in familial amyotrophic lateral sclerosis (ALS) and multisystem proteinopathoy (MSP) are all involved in the fibril core and contribute to fibril stability. Our work illuminates structural understandings of the pathological amyloid aggregation of hnRNPA1 and the amyloid disaggregase activity of Kapβ2, and highlights the multiple roles of PY-NLS in hnRNPA1 homeostasis.