N-terminal acetylation modestly enhances phase separation and reduces aggregation of the low-complexity domain of RNA-binding protein fused in sarcoma

Abstract

The RNA-binding protein fused in sarcoma (FUS) assembles via liquid-liquid phase separation (LLPS) into functional RNA granules and aggregates in amyotrophic lateral sclerosis associated neuronal inclusions. Several studies have demonstrated that posttranslational modification (PTM) can significantly alter FUS phase separation and aggregation, particularly charge-altering phosphorylation of the nearly uncharged N-terminal low complexity domain of FUS (FUS LC). However, the occurrence and impact of N-terminal acetylation on FUS phase separation remains unexplored, even though N-terminal acetylation is the most common PTM in mammals and changes the charge at the N-terminus. First, we find that FUS is predominantly acetylated in two human cell types and stress conditions. Next, we show that recombinant FUS LC can be acetylated when co-expressed with the NatA complex in Escherichia coli. Using NMR spectroscopy, we find that N-terminal acetylated FUS LC (FUS LC Nt-Ac) does not notably alter monomeric FUS LC structure or motions. Despite no difference in structure, Nt-Ac-FUS LC phase separates more avidly than unmodified FUS LC. More importantly, N-terminal acetylation of FUS LC reduces aggregation. Our findings highlight the importance of N-terminal acetylation of proteins that undergo physiological LLPS and pathological aggregation.

Keywords: Amber99SBws-STQ; aggregation; coarse-grained modeling; intrinsically disordered protein; liquid-liquid phase separation; molecular simulation; nuclear magnetic resonance; posttranslational modification.

FIGURE 1

FUS LC is N‐terminally acetylated in human cells and in Escherichia coli expressing NatA. (a) FUS domain architecture including the low‐complexity N‐terminal domain (FUS LC). (b) N‐terminal peptides identified from mass spectrometry of FUS immunoprecipitates from human cell lines HEK293T or H4 cell lines under control (DMSO) or cellular stress conditions as labeled. All N‐terminal peptides (except ones in black) show removal of Met1 and acetylation at Ala2. (c) Deconvoluted mass spectrum of recombinant FUS LC co‐expressed with NatA complex in E. coli. At these conditions, several N‐terminal aminopeptidase products are observed (e.g., FUS LC 2–163 + His‐tag, FUS LC 3–163 + His‐tag) which all show N‐terminal acetylation of resulting N‐terminal amino acid (see Table 1). The acetylated protein is lower in apparent ion counts (y‐axis) likely because of IPTG induction at higher than optimal OD (See Figure S2), though we note that removing the charged N‐terminus may decrease ionization efficiency and make total ion count an unreliable estimate of abundance. FUS, fused in sarcoma; FUS LC, low complexity domain of FUS; OD, optical density

FIGURE 2

N‐terminal acetylation does not change disordered structure of FUS LC. (a) ¹H‐¹⁵N HSQC NMR spectrum of N‐terminally acetylated FUS LC (red) compared to a ¹H‐¹⁵N HSQC spectrum of unacetylated FUS LC (black). Despite some small apparent chemical shifts changes, the narrow spectrum suggests FUS LC remains intrinsically disordered after N‐terminal acetylation. Additional resonances (A2, S3, N4, D5) are visible in the acetylated spectrum due to removal of the basic amine that broadens these resonances in unacetylated FUS LC. Note that A2 is aliased (appears near 110 ppm instead of below 125 ppm) because of the narrow ¹⁵N spectral width chosen. (b) Small N‐terminal region ¹⁵N and ¹H chemical shift perturbations are observed for Nt‐Ac FUS LC compared to unacetylated FUS LC. (c) Secondary shifts of the ¹³Cα and ¹³Cβ for the unacetylated and acetylated FUS LC are indistinguishable. (d) Structural ensembles from all atom simulations of FUS LC 2–44 show some predicted α‐helical or 3₁₀ helical secondary structure but show no evidence for helical enhancement caused by acetylation. FUS, fused in sarcoma; FUS LC, low complexity domain of FUS

FIGURE 3

Effect of N‐terminal acetylation on FUS LC motions. (a) NMR diffusion measurements show that Nt‐Ac FUS LC has a slightly larger apparent radius of hydration, R _h (nm), compared to unacetylated FUS LC. Lysozyme is used as a reference standard. (b) Radius of gyration, R _g, from all atom molecular simulations of the shorter peptide FUS LC 2–44 show no significant effect of acetylation. (c) NMR spin relaxation parameters ¹⁵N R ₂, ¹⁵N R ₁, and heteronuclear NOE show slight differences in N‐terminal region of Nt‐Ac FUS LC. Each dataset was plotted as a mean of ±SD. FUS, fused in sarcoma; FUS LC, low complexity domain of FUS

FIGURE 4

N‐terminal acetylation slightly promotes phase separation of FUS LC. (a) Amount of protein remaining in the supernatant as a function of increasing NaCl concentration after phase separated droplets are spun out shows N‐terminal acetylation decreases the apparent saturation concentration for FUS LC LLPS. Representative experiment of three biological replicates are plotted as mean of ±SD of technical replicates. Scale bars are 50 μm. (b) DIC microscopy of unacetylated and Nt‐Ac FUS LC show both forms of FUS LC form liquid droplets. (c) Phase diagram for FUS LC LLPS from coarse‐grained molecular dynamics simulations of Nt‐Ac FUS LC (red) and unacetylated FUS LC (black) show slight differences in saturation concentration (i.e., the left arm of the phase diagram, see inset) and condensed phase concentration (i.e., right arm of the phase diagram). (inset) Phase diagram in log scale of concentration which shows differences in saturation concentration. FUS, fused in sarcoma; FUS LC, low complexity domain of FUS; LLPS, liquid–liquid phase separation

FIGURE 5

N‐terminal acetylation slows FUS LC aggregation in vitro. FUS LC aggregation was observed after LLPS induction in either no salt or 150 mM NaCl with either quiescent (a), rocking (b), or shaking at 1200 rpm in a ThermoMixer (c). Large aggregates formed in the ThermoMixer samples and were seen after 1 hr for both FUS LC and Nt‐Ac FUS LC. Largest aggregates were found in samples containing salt. Droplets remained even at the 24‐hr time point for Nt‐Ac FUS LC at gently rocked conditions while irregularly shaped aggregates were seen as early as 4 hr for the unacetylated FUS LC. Scale bars are 50 μm. FUS, fused in sarcoma; FUS LC, low complexity domain of FUS; LLPS, liquid–liquid phase separation

FIGURE 6

FUS LC phase separation and interactions impacted by N‐terminal acetylation. FUS LC is sufficient for phase separation via weak, multivalent contacts between the repetitive polar residue sequence motifs present in the sequence. Acetylation (bottom left) removes the positive charge of the N‐terminus. N‐terminal acetylation of FUS LC promotes phase separation but disrupts aggregation. FUS, fused in sarcoma; FUS LC, low complexity domain of FUS