Molecular Details of Protein Condensates Probed by Microsecond Long Atomistic Simulations

Abstract

The formation of membraneless organelles in cells commonly occurs via liquid-liquid phase separation (LLPS) and is in many cases driven by multivalent interactions between intrinsically disordered proteins (IDPs). Investigating the nature of these interactions, and their effect on dynamics within the condensed phase, is therefore of critical importance but very challenging for either simulation or experiment. Here, we study these interactions and their dynamics by pairing a novel multiscale simulation strategy with microsecond all-atom MD simulations of a condensed, IDP-rich phase. We simulate two IDPs this way, the low complexity domain of FUS and the N-terminal disordered domain of LAF-1, and find good agreement with experimental information about average density, water content, and residue-residue contacts. We go significantly beyond what is known from experiments by showing that ion partitioning within the condensed phase is largely driven by the charge distribution of the proteins and-in the cases considered-shows little evidence of preferential interactions of the ions with the proteins. Furthermore, we can probe the microscopic diffusive dynamics within the condensed phase, showing that water and ions are in dynamic equilibrium between dense and dilute phases, and their diffusion is reduced in the dense phase. Despite their high concentration in the condensate, the protein molecules also remain mobile, explaining the observed liquid-like properties of this phase. We finally show that IDP self-association is driven by a combination of nonspecific hydrophobic interactions as well as hydrogen bonds, salt bridges, and π-π and cation-π interactions. The simulation approach presented here allows the structural and dynamical properties of biomolecular condensates to be studied in microscopic detail and is generally applicable to single- and multicomponent systems of proteins and nucleic acids involved in LLPS.

Figure 1:

Procedure to set up all-atom simulations of dense phase. (a) An initial configuration is generated by CG simulations in a box with elongated z-dimension. (b) All-atom coordinates reconstructed from CG C_α coordinates templates for each chain using PDB database. (c) Reconstructed chains reassembled into condensed phase and sidechain clashes relieved using short Monte Carlo simulations with frozen backbone. (d) Addition of solvent and ions to generate complete system at all-atom resolution.

Figure 2:

Density profiles from all-atom slab simulations of FUS LC (left) and LAF-1 RGG (right). Components are shown in the legend. Dashed lines in (a) indicate experimentally determined values and dashed lines in (c) and (d) indicate the predicted ion concentration using concentrations of protein cationic and anionic residues.

Figure 3:

Protein self-diffusion coefficients within the slab along the z-axis, from all-atom slab simulations of FUS LC (blue) and LAF-1 RGG (red) are shown as a function of the lag time. Dashed horizontal line indicates experimentally determined diffusivity value for FUS LC.

Figure 4:

Intermolecular contacts within the condensed phase of FUS LC (left) and LAF-1 RGG (right), as a function of residue index (a and b) and amino acid types (c and d). The intermolecular contacts normalized by the relative abundance of each amino acid in the sequence are shown in e and f. In each of the figure, the bottom panel shows the one dimensional summation. The black dashed lines in a and b show the position of Tyr residues. The blues lines in c and d show the number of amino acids (N_AA) in the sequence.

Figure 5:

Interaction modes contributing to the intermolecular contacts separated into contributions between backbone atoms (bb-bb, a and b), between backbone and sidechain atoms (bb-sc, c and d) and between sidechain atoms (sc-sc, e and f). The amino acid pairs are sorted by the number of contacts formed between these pairs in each group as shown in Fig. S11. Some configurations of representative interacting amino acids are shown in g for FUS LC Tyr:Tyr, h for FUS LC Tyr:Gln, i for LAF-1 RGG Tyr:Arg and j for LAF-1 RGG Asp:Arg. The configurations are aligned to Tyr in g, h and i and to Asp in j. The color code is the same as the interaction mode shown in the legend. Rings are shown in paperchain representation.