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. 2020 Oct 15;25(20):4705.
doi: 10.3390/molecules25204705.

Expansion of Intrinsically Disordered Proteins Increases the Range of Stability of Liquid-Liquid Phase Separation

Adiran Garaizar  1 , Ignacio Sanchez-Burgos  1 , Rosana Collepardo-Guevara  1   2   3 , Jorge R Espinosa  1
Affiliations

Expansion of Intrinsically Disordered Proteins Increases the Range of Stability of Liquid-Liquid Phase Separation

Adiran Garaizar et al. Molecules. .

Abstract

Proteins containing intrinsically disordered regions (IDRs) are ubiquitous within biomolecular condensates, which are liquid-like compartments within cells formed through liquid-liquid phase separation (LLPS). The sequence of amino acids of a protein encodes its phase behaviour, not only by establishing the patterning and chemical nature (e.g., hydrophobic, polar, charged) of the various binding sites that facilitate multivalent interactions, but also by dictating the protein conformational dynamics. Besides behaving as random coils, IDRs can exhibit a wide-range of structural behaviours, including conformational switching, where they transition between alternate conformational ensembles. Using Molecular Dynamics simulations of a minimal coarse-grained model for IDRs, we show that the role of protein conformation has a non-trivial effect in the liquid-liquid phase behaviour of IDRs. When an IDR transitions to a conformational ensemble enriched in disordered extended states, LLPS is enhanced. In contrast, IDRs that switch to ensembles that preferentially sample more compact and structured states show inhibited LLPS. This occurs because extended and disordered protein conformations facilitate LLPS-stabilising multivalent protein-protein interactions by reducing steric hindrance; thereby, such conformations maximize the molecular connectivity of the condensed liquid network. Extended protein configurations promote phase separation regardless of whether LLPS is driven by homotypic and/or heterotypic protein-protein interactions. This study sheds light on the link between the dynamic conformational plasticity of IDRs and their liquid-liquid phase behaviour.

Keywords: biological phase transitions; computer simulations; proteins.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Minimal IDR coarse-grained model to investigate the impact of the conformational ensemble in LLPS. (A) Illustration of the coarse-grained protein model developed in this work with one bead representing a group of amino acids. The excluded volume interactions among beads are depicted with grey dashed circles, and the multivalent attracting interactions with yellow dashed lines. (B) Representative snapshots of the different types of IDRs studied. (i) Intrinsically disordered protein of N = 20. (ii) Intrinsically disordered protein of N = 80. (iii) Semi-compact globular protein of N = 20. (iv) Compact globular protein of N = 20. (v) Compact globular protein of N = 80. (C) Schematic representation of the impact of the angular constant (Kθ) in the representative configuration of the coarse-grained IDRs of N = 20. Higher Kθ implies conformational landscapes enriched in extended states while lower ones enhance the emergence of structures preferentially collapsed. (D) Snapshot of a direct coexistence simulation in which LLPS is driven by homotypic interactions. (E) Sketch of a binary mixture direct coexistence simulation where LLPS is driven by heterotypic interactions, phase separation takes place in presence of a high affinity partner. Multivalent interactions between proteins of the different type (heterotypic) are twice more attractive (2ϵ) than those among proteins of the same type (ϵ).
Figure 2
Figure 2
Direct coexistence simulation box of: (A) 128 interacting globular proteins of length N = 80 at T*=2.5. (B) 512 intrinsically disordered proteins of length N = 20 at T*=2.25. Individual proteins are depicted by different colours.
Figure 3
Figure 3
Conformational landscape and phase diagram of the different 20-bead IDRs. (A) Probability histograms of the radius of gyration of the different IDRs at T*=0.65Tc,FE*. Tc,FE* refers to the critical temperature of the fully extended IDR, Tc,FE* = 2.76, which is the highest critical temperature of the four different IDRs. Results for the fully flexible random coil (Kθ* = 0) are depicted in purple, while those ones for the lightly (Kθ* = 1), moderately (Kθ* = 3) and fully extended (Kθ* = 20) IDRs in pink, green and red, respectively. Solid lines represent the radius of gyration distribution for proteins that form part of the condensed liquid phase, whereas dashed lines account for the radius of gyration distribution of proteins belonging to the diluted liquid phase. (B) Liquid-liquid coexistence lines in the T*−ρ* plane for the fully flexible random coil (purple), lightly extended (pink), moderately extended (green) and fully extended (red) IDRs. The temperature is normalized by the critical temperature of the fully extended IDR, Tc,FE*. Filled circles account for the coexisting densities computed via DC simulations and empty ones for the estimation of the critical points using Equations (4) and (5). The horizontal black dashed line represents the temperature at which the radius of gyration probability histograms in panel A were evaluated. (C) Mean value of the radius of gyration (<Rg*>) as a function of the renormalized temperature, T*/Tc,FE*, for the previously shown IDRs. The same colour code as in panel A and B applies here. The values of Rg* in the condensed phase along the coexisting densities are depicted by empty diamonds whereas filled circles represent the same but for IDRs in the protein-poor liquid phase.
Figure 4
Figure 4
Liquid-network connectivity explains LLPS. Average number of inter-molecular contacts per protein in the condensed liquid phase, NInterC, as a function of T*/Tc,FE* for the different 20-bead proteins described in the legend.
Figure 5
Figure 5
Impact of the liquid–liquid phase transition on the protein conformational ensemble. Boxplots of the radius of gyration vs. normalised temperature (T*/Tc,FE*) for the fully flexible random coil, and the lightly and moderately extended N = 20 IDRs. Orange boxplots account for Rg* in the diluted phase while blue ones for IDRs in the condensed phase. Fully extended IDR boxplots have not been included since Rg* is not different in both phases (according to the Kolmogorov-Smirnov test [115]) and does not vary with temperature either as shown in Figure 3C. Boxplots are a 5 number summary of the radius of gyration distribution. The box bounds represent the first and the third quartile of the histograms and the line intersecting the box is the median. The whiskers represent the maximum and minimum values of the histograms.
Figure 6
Figure 6
Role of globular and compact conformations in LLPS. (A) Phase diagram of different conformational ensembles of the 20-bead sequence. Results for the random coil are depicted in purple, for the fully extended IDR in red, while for the semi-compact and compact globular proteins in brown and grey, respectively. Filled circles represent the liquid coexisting densities evaluated by means of DC simulations, empty symbols the estimated critical point for each system via Equations (4) and (5), and continuous lines are included as a guide for the eye. (B) Phase diagram of an 80-bead fully flexible random coil with <Rg> = 4.42σ (at T*/Tc,RC*=0.9) (blue) and a collapsed globular conformation with <Rg> = 2.45σ (red) of length N = 80. Note that in both panels, temperature is normalised by the critical temperature of the conformational ensemble with highest critical point for each length, Tc,FE* = 2.76 for the fully extended 20-bead IDR and Tc,RC* = 2.68 for the random coil of 80 beads. The structured globular domains for which the phase diagram is evaluated here are shown in Figure 1.
Figure 7
Figure 7
Impact of the conformational plasticity in heterotypically driven LLPS (A) Liquid-liquid coexistence lines of two 50:50 binary mixtures of 20-bead IDRs with different conformational ensembles: filled black circles represent the coexistence densities for a mixture of fully flexible random coils while blue circles for a mixture of moderately extended IDRs. Empty circles indicate the estimated critical point of each mixture. Note that temperature is renormalized by the critical temperature of the moderately extended IDR binary mixture, Tc,ME* = 3.8. (B) Average radius of gyration, <Rg*> of the IDRs as a function of the renormalized temperature, T*/Tc,ME*. Circles indicate <Rg*> in the diluted liquid phase and diamonds inside the condensed one. The same colour code of the aside panel applies here.
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