Subcellular membrane-less assemblies are a reinvigorated area study in biology with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces we determined atomic structures of five segments of protein low-complexity domains associated with membrane-less assemblies. Their common structural feature is the stacking of segments into kinked β-sheets which pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic sidechains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, known to form networks and localize to membrane-less assemblies.

The normally soluble TAR DNA Binding Protein 43 (TDP-43) is found aggregated both in reversible stress granules and irreversible pathogenic amyloid. In TDP-43, the low complexity domain (LCD) is believed to be involved in both types of aggregation. To discover the structural origins of these two modes of β-sheet rich aggregation, we have determined ten structures of segments of the LCD of human TDP-43. Six of these segments form steric zippers characteristic of the spines of pathogenic amyloid fibrils; four others form LARKS, the labile amyloid-like interactions characteristic of protein hydrogels and proteins found in membrane-less organelles, including stress granules. Supporting a hypothetical pathway from reversible to irreversible amyloid aggregation, we found that familial ALS variants of TDP-43 convert LARKS to irreversible aggregates. Our structures suggest how TDP-43 adopts both reversible and irreversible β-sheet aggregates, and the role of mutation in the possible transition of reversible to irreversible pathogenic aggregation.

Aggregated Tau protein is associated with over 20 neurological disorders including Alzheimer’s disease. Previous work has shown that Tau’s sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length Tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of Tau aggregation. Two structures of this segment determined by the cryo EM method MicroED explain its dominant influence on Tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit Tau aggregation but also inhibit the ability of exogenous full-length Tau fibrils to seed intracellular Tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of Tau that may account for a subset of prion-like strains of Tau.

The DNA/RNA processing protein TDP-43 undergoes both functional and pathogennic aggregation. Functional TDP-43 aggregates form reversible, transient species such as nuclear bodies, stress granules, and myo-granules. Pathogenic, irreversible TDP-43 aggregates form in amyotrophic lateral sclerosis (ALS) and other neurodegenerative conditions. Here we find the features of TDP-43 fibrils that confer both reversibility and irreversibility by determining structures of two segments reported to be the pathogenic cores of human TDP-43 aggregation: SegA (residues 311-360), which forms three polymorphs, all with dagger-shaped folds; and SegB A315E (residues 286-331 containing the ALS hereditary mutation A315E), which forms Rshaped folds. Energetic analysis suggests that the dagger-shaped polymorphs represent irreversible fibril structures, whereas the SegB polymorph may participate in both reversible and irreversible fibrils. Our structures reveal the polymorphic nature of TDP-43 and suggest how the A315E mutation converts the R-shaped polymorph to an irreversible form which enhances pathology.