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[Preprint]. 2024 Feb 15:2024.02.15.580391.
doi: 10.1101/2024.02.15.580391.

Expanding the molecular grammar of polar residues and arginine in FUS prion-like domain phase separation and aggregation

Noah Wake  1 , Shuo-Lin Weng  2 , Tongyin Zheng  3 , Szu-Huan Wang  3 , Valentin Kirilenko  3 , Jeetain Mittal  2   4   5 , Nicolas L Fawzi  3
Affiliations

Expanding the molecular grammar of polar residues and arginine in FUS prion-like domain phase separation and aggregation

Noah Wake et al. bioRxiv. .

Abstract

A molecular grammar governing low-complexity prion-like domains phase separation (PS) has been proposed based on mutagenesis experiments that identified tyrosine and arginine as primary drivers of phase separation via aromatic-aromatic and aromatic-arginine interactions. Here we show that additional residues make direct favorable contacts that contribute to phase separation, highlighting the need to account for these contributions in PS theories and models. We find that tyrosine and arginine make important contacts beyond only tyrosine-tyrosine and tyrosine-arginine, including arginine-arginine contacts. Among polar residues, glutamine in particular contributes to phase separation with sequence/position-specificity, making contacts with both tyrosine and arginine as well as other residues, both before phase separation and in condensed phases. For glycine, its flexibility, not its small solvation volume, favors phase separation by allowing favorable contacts between other residues and inhibits the liquid-to-solid (LST) transition. Polar residue types also make sequence-specific contributions to aggregation that go beyond simple rules, which for serine positions is linked to formation of an amyloid-core structure by the FUS low-complexity domain. Hence, here we propose a revised molecular grammar expanding the role of arginine and polar residues in prion-like domain protein phase separation and aggregation.

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Figures

Figure 1.
Figure 1.. Phase separation of FUS LC-RGG1
a FUS domain structure and residue composition of LC-RGG1, showing distinct composition of LC and RGG1 b Salt dependence of phase separation (i.e. the saturation concentration) compared for FUS LC and FUS LC-RGG1, measured by the amount of protein remaining in the supernatant. Insert: schematic of the centrifugation experiment to measure the saturation concentration. c The average intramolecular distance Rij between the ith and jth residues, calculated from atomistic simulations of isolated monomers (“single chains”) of FUS wild type LC-RGG1, LC, and RGG1. Standard error of the mean is computed over 3 independent trajectories. The dashed lines are the fitting curves using the following function: Rij=b∣i-j∣. d Intramolecular interaction profiles calculated from atomistic simulations of FUS wild type LC-RGG1, LC, and RGG1 single chains, binned by residue position. The left column shows the one-dimensional summation of LC-RGG1 contacts, and the bottom row shows the one-dimensional summation of individual LC and RGG1 contacts. e Motions in the condensed phase assessed by 15N NMR spin relaxation R1, R2, and heteronuclear NOE. Sequence regions of depressed R2 relaxation are highlighted (grey boxes) and overlayed with possible amyloid forming cores (black boxes). Average relaxation parameters for LC and RGG1 domains in the condensed phase are indicated (grey dashed line) and show that the RGG1 domains show faster reorientational motions than the LC.
Figure 2.
Figure 2.. LC-RGG1 makes both homotypic and heterotypic contacts in the LC-RGG1 phase, including aromatic and arginine residues as well as polar residues
a Schematic of the 13C- (left) and 15N- (right) edited (double 13C/15N) filtered NOE-based NMR experiments used to measure only intermolecular contacts formed in the LC-RGG1 condensed phase. b 13C-edited intermolecular NOEs in the LC-RGG1 phase show many residue types have contacts, including LC:LC, LC:RGG1, and RGG1:RGG1 (see Fig. S2a for complete set of observed NOEs). NOE intensities are corrected for (small) intramolecular contributions by subtracting intensities measured in natural abundance control samples (see Methods), and presented as stacked bars for different resolved positions in each residue type. NOEs to aromatic residue positions are measured in a separate experiment and intensities cannot directly be compared to other positions so are presented separated by a dashed line (see Methods). c 15N-edited intermolecular NOEs show that Gln, Tyr, and Arg intermolecular contacts span across the entire LC-RGG1 sequence, including to side chain Arg and Gln/Asn positions. (see Fig. S2b for complete set of observed NOEs) d Interaction profiles calculated from atomistic simulations of FUS wild type LC-RGG1 and LC condensed phase (slab simulations), binned by residue position. The left column shows the one-dimensional summation of LC-RGG1 contacts, and the bottom row shows the one-dimensional summation of LC contacts. e The normalized numbers of contacts (by numbers of residues) binned by residue type, calculated from atomistic simulations of FUS wild type LC-RGG1 condensed phase. Inset: Correlation between the pairwise contacts in the LC condensed phased compared to the LC-RGG1 condensed phase. f Molecular images of the LC and RGG1 contacts formed in the LC-RGG1 condensed phase simulations, highlighting the different types of residue pair contacts formed in the phase.
Figure 3.
Figure 3.. Contacts between tyrosine residues and other polar residues are present in the dispersed phase as well as the condensed phase
a Schematic of the samples utilized for intermolecular aromatic 13C-edited filtered NOE NMR experiments in the dispersed phase for high concentration FUS LC 8Y→S samples. b Using a phase separation deficient FUS LC variant, 8Y→S, intermolecular NOE experiments demonstrate contacts formed between Tyrosine and Glutamine (red, see dashed lines), which are also observed in the condensed phase of FUS LC wild type (green). Using a FUS LC mutant in which i+1/−1 Y/Q sequence pairs are removed, we see that these same interaction are observed in a 13C-edited intramolecular NOE experiment for intramolecular contacts. Control samples made with 100% 13C-15N isotopically labeled (blue) and 100% natural abundance (purple) FUS LC 8Y→S were included as controls to confirm the presence of intermolecular contacts in the mixed sample (red) are bona fide intermolecular NOEs. c The correlations between the numbers of contacts formed in the single chain (x-axis) and condensed phase (y-axis) simulations, separated into LC homotypic, RGG1 homotypic, and heterotypic contacts.
Figure 4.
Figure 4.. Polar residues alter phase equilibria based on their identity and distribution across the sequence.
a Schematic of the sequence position of variants used in this study. Complete replacement substitutions were only made in the LC domain of FUS. b Measurements of the Csat for FUS LC or FUS LC-RGG1 variants in comparison to the wild type. Hatched bar indicates no phase separation. FUS LC experiments were performed at 300 μM and LC-RGG1 at 60 μM in 150 mM NaCl, 20 mM HEPES, pH 7.0 at ambient temperature. c DIC micrographs of the substitution variants in the same buffer conditions. d Schematic of the FUS LC variants partially modifying polar residues at 4 QQ motifs or 12 S positions. White bars represent which serine or glutamine residues were replaced. e Quantification of the effects on the Csat of the FUS LC variants vs. the wild type sequence conducted at 300 μM FUS LC in 20 mM HEPES, pH 7.0. f Correlation plot between the observed Csat for each residue type at 150 mM NaCl condition. g 1D profiles of the condensed phase simulation contacts for the wild type and S→G LC mutants, showing higher contacts for residues adjacent to positions where serine to glycine substitutions (dashed lines) were made. h Correlation between the number of pairwise contacts formed for the wild type versus the S→G sequence in the simulated condensed phases, showing higher contacts for many pairs including QY and YY in the S→G and fewer contacts for the positions that were converted from serine to glycine (red dots).
Figure 5.
Figure 5.. Polar residue identity contributes to the liquid-to-solid transition
a Schematics depicting the method of inducing aggregation for the microscopy-based experiment and the ThT based assay. ThT enhancement curves (blue) are fit with a sigmoid (red) and both the ThT enhancement transition and maximum ThT intensity are determined from the fit. b Time until ThT positive transition (longer bars indicate ThT-detected aggregation) and ThT intensity at 24 hours (larger bars indicate more ThT enhancement) of the variants in the LC domain when exposed to either quiescent and double orbital shaking conditions. c DIC micrographs of the variants in the LC and LC-RGG1 when subjected to 24 hours of quiescent and orbital shaking conditions (150 rpm, up to 24 hours) d Same as in b but for the variants in the LC-RGG1 sequence (24 hour duration, double orbital shaking). e Same as in c but for the variants in the LC-RGG1 sequence under quiescent or 150 rpm shaking conditions and up to 24 hours.
Figure 6.
Figure 6.. Serine side chain specifically contributes to formation of aggregates from FUS LC droplets
a Time until ThT positive transition and ThT fluorescence at 24 hours of FUS LC variants with partial polar residue substitution when subjected to either quiescent or aggregation-inducing conditions (double orbital shaking). b DIC micrographs of FUS LC variants with partial polar residue substitution when subjected to quiescent or aggregation-inducing mutations (150 rpm shaking). c Schematic of the serine to alanine sequences generated to determine which serines contribute most significantly to aggregation in FUS LC. d same as a but for the 4S→A variants under quiescent or aggregating conditions (double orbital shaking) to determine which subset of the 12 S→A serines are most important for aggregation e same as b but for the 4S→A variants under quiescent or aggregating conditions (150 rpm shaking) f Serines modified in this study mapped to their location in a proposed amyloid core (PDB:5W3N) for serines in 4S→A#1 (red) and 4S→A#2 (green). Glutamine residues identified as forming hydrogen bonds in the structure (gray sticks) are found in 4S→A#1.
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