Mechanism of karyopherin-ß2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X).

Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid-liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-ß2 (Kapß2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapß2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapß2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapß2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapß2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapß2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.

Molecular structure and interactions within amyloid-like fibrils formed by a low-complexity protein sequence from FUS.

Protein domains without the usual distribution of amino acids, called low complexity (LC) domains, can be prone to self-assembly into amyloid-like fibrils. Self-assembly of LC domains that are nearly devoid of hydrophobic residues, such as the 214-residue LC domain of the RNA-binding protein FUS, is particularly intriguing from the biophysical perspective and is biomedically relevant due to its occurrence within neurons in amyotrophic lateral sclerosis, frontotemporal dementia, and other neurodegenerative diseases. We report a high-resolution molecular structural model for fibrils formed by the C-terminal half of the FUS LC domain (FUS-LC-C, residues 111-214), based on a density map with 2.62 Å resolution from cryo-electron microscopy (cryo-EM). In the FUS-LC-C fibril core, residues 112-150 adopt U-shaped conformations and form two subunits with in-register, parallel cross-ß structures, arranged with quasi-21 symmetry. All-atom molecular dynamics simulations indicate that the FUS-LC-C fibril core is stabilized by a plethora of hydrogen bonds involving sidechains of Gln, Asn, Ser, and Tyr residues, both along and transverse to the fibril growth direction, including diverse sidechain-to-backbone, sidechain-to-sidechain, and sidechain-to-water interactions. Nuclear magnetic resonance measurements additionally show that portions of disordered residues 151-214 remain highly dynamic in FUS-LC-C fibrils and that fibrils formed by the N-terminal half of the FUS LC domain (FUS-LC-N, residues 2-108) have the same core structure as fibrils formed by the full-length LC domain. These results contribute to our understanding of the molecular structural basis for amyloid formation by FUS and by LC domains in general.

Side Chain Hydrogen-Bonding Interactions within Amyloid-like Fibrils Formed by the Low-Complexity Domain of FUS: Evidence from Solid State Nuclear Magnetic Resonance Spectroscopy.

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-ß 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.

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.