In aqueous solutions, the 214-residue low-complexity domain of the FUS protein (FUS-LC) is known to undergo liquid-liquid phase separation and also to self-assemble into amyloid-like fibrils. In previous work based on solid state nuclear magnetic resonance (ssNMR) methods, a structural model for the FUS-LC fibril core was developed, showing that residues 39-95 form the fibril core. Unlike fibrils formed by amyloid-β peptides, α-synuclein, and other amyloid-forming proteins, the FUS-LC core is largely devoid of purely hydrophobic amino acid side chains. Instead, the core-forming segment contains numerous hydroxyl-bearing residues, including 18 serines, six threonines, and eight tyrosines, suggesting that the FUS-LC fibril structure may be stabilized in part by inter-residue hydrogen bonds among side chain hydroxyl groups. Here we describe ssNMR measurements, performed on ²H,¹⁵N,¹³C-labeled FUS-LC fibrils, that provide new information about the interactions of hydroxyl-bearing residues with one another and with water. The ssNMR data support the involvement of specific serine, threonine, and tyrosine residues in hydrogen-bonding interactions. The data also reveal differences in hydrogen exchange rates with water for different side chain hydroxyl groups, providing information about solvent exposure and penetration of water into the FUS-LC fibril core.

Most, if not all, peptide- and protein-based hydrogels formed by self-assembly can be characterized as kinetically trapped 3D networks of fibrils. The propensity of disease-associated amyloid-forming peptides and proteins to assemble into polymorphic fibrils suggests that cross-β fibrils comprising hydrogels may also be polymorphic. We use solid-state NMR to determine the molecular and supramolecular structure of MAX1, a de novo designed gel-forming peptide, in its fibrillar state. We find that MAX1 adopts a β-hairpin conformation and self-assembles with high fidelity into a double-layered cross-β structure. Hairpins assemble with an in-register Syn orientation within each β-sheet layer and with an Anti orientation between layers. Surprisingly, although the MAX1 fibril network is kinetically trapped, solid-state NMR data show that fibrils within this network are monomorphic and most likely represent the thermodynamic ground state. Intermolecular interactions not available in alternative structural arrangements apparently dictate this monomorphic behavior.

Human genetic studies have given evidence of familial, disease-causing mutations in the analogous amino acid residue shared by three related RNA binding proteins causative of three neurological diseases. Alteration of aspartic acid residue 290 of hnRNPA2 to valine is believed to predispose patients to multisystem proteinopathy. Mutation of aspartic acid 262 of hnRNPA1 to either valine or asparagine has been linked to either amyotrophic lateral sclerosis or multisystem proteinopathy. Mutation of aspartic acid 378 of hnRNPDL to either asparagine or histidine has been associated with limb girdle muscular dystrophy. All three of these aspartic acid residues map to evolutionarily conserved regions of low-complexity (LC) sequence that may function in states of either intrinsic disorder or labile self-association. Here, we present a combination of solid-state NMR spectroscopy with segmental isotope labeling and electron microscopy on the LC domain of the hnRNPA2 protein. We show that, for both the wild-type protein and the aspartic acid 290-to-valine mutant, labile polymers are formed in which the LC domain associates into an in-register cross-β conformation. Aspartic acid 290 is shown to be charged at physiological pH and immobilized within the polymer core. Polymers of the aspartic acid 290-to-valine mutant are thermodynamically more stable than wild-type polymers. These observations give evidence that removal of destabilizing electrostatic interactions may be responsible for the increased propensity of the mutated LC domains to self-associate in disease-causing conformations.

Polymerization and phase separation of proteins containing low-complexity (LC) domains are important factors in gene expression, mRNA processing and trafficking, and localization of translation. We have used solid-state nuclear magnetic resonance methods to characterize the molecular structure of self-assembling fibrils formed by the LC domain of the fused in sarcoma (FUS) RNA-binding protein. From the 214-residue LC domain of FUS (FUS-LC), a segment of only 57 residues forms the fibril core, while other segments remain dynamically disordered. Unlike pathogenic amyloid fibrils, FUS-LC fibrils lack hydrophobic interactions within the core and are not polymorphic at the molecular structural level. Phosphorylation of core-forming residues by DNA-dependent protein kinase blocks binding of soluble FUS-LC to FUS-LC hydrogels and dissolves phase-separated, liquid-like FUS-LC droplets. These studies offer a structural basis for understanding LC domain self-assembly, phase separation, and regulation by post-translational modification.

Low complexity (LC) head domains 92 and 108 residues in length are, respectively, required for assembly of neurofilament light (NFL) and desmin intermediate filaments (IFs). As studied in isolation, these IF head domains interconvert between states of conformational disorder and labile, β-strand-enriched polymers. Solid-state NMR (ss-NMR) spectroscopic studies of NFL and desmin head domain polymers reveal spectral patterns consistent with structural order. A combination of intein chemistry and segmental isotope labeling allowed preparation of fully assembled NFL and desmin IFs that could also be studied by ss-NMR. Assembled IFs revealed spectra overlapping with those observed for β-strand-enriched polymers formed from the isolated NFL and desmin head domains. Phosphorylation and disease-causing mutations reciprocally alter NFL and desmin head domain self-association yet commonly impede IF assembly. These observations show how facultative structural assembly of LC domains via labile, β-strand-enriched self-interactions may broadly influence cell morphology.