Single-chain and condensed-state behavior of hnRNPA1 from molecular simulations

Abstract

Intrinsically disordered proteins (IDPs) are essential components for the formation of membraneless organelles, which play key functional and regulatory roles within biological systems. These complex assemblies form and dissolve spontaneously over time via liquid-liquid phase separation of IDPs. Mutations in their amino acid sequence can alter their phase behavior, which has been linked to the emergence of severe diseases. We study the conformation and phase behavior of a low-complexity domain of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), using coarse-grained implicit solvent molecular simulations. We systematically analyze how these properties are affected by the number of aromatic residues within the examined sequences. We find a significant compaction of the chains and an increase in the critical temperature with increasing number of aromatic residues. The local persistence length is determined in single-chain simulations, revealing strong sequence-specific variations along the chain contour. Comparing single-chain and condensed state simulations, we find much more collapsed polymer conformations in the dilute systems, even at temperatures near the estimated θ-temperature of the solution. These observations strongly support the hypothesis that aromatic residues play a dominant role for condensation, which is further corroborated by a detailed analysis of the intermolecular contacts, and conversely that important properties of condensates are captured in coarse-grained simulations. Interestingly, we observe density inhomogeneities within the condensates near criticality, which are driven by electrostatic interactions. Finally, we find that the relatively small fraction of hydrophobic residues in the IDPs results in interfacial tensions which are significantly lower compared to typical combinations of immiscible simple liquids.

Effect of Charge Distribution on the Dynamics of Polyampholytic Disordered Proteins

“…We excluded pairs of residues that were separated by one or two bonds from the map because their contacts were primarily determined by the chain connectivity. The purpose of showing both the distance-based contact maps and energy maps is that they are complementary but distinct metrics, and using both is especially helpful for reliably determining contacts between residues of different sizes . As seen from Figures and S3, contacts were more evenly distributed along the chain contour for more uniformly charge-patterned sequences with nSCD ≲ 0.035 (EKV1 to EKV3).…”

Conformational Properties of Polymers at Droplet Interfaces as Model Systems for Disordered Proteins

“…Membraneless organelles or biomolecular condensates formed through liquid – liquid phase separation (LLPS) have been widely reported in various cellular functions, including gene expression, signal transduction, stress response, and the assembly of macromolecular complexes. − Examples of such condensates include the nucleolus, Cajal bodies, P bodies, and stress granules. Intrinsically disordered proteins (IDPs) play an important role in the formation of biomolecular condensates through LLPS. − Due to the numerous similarities between IDPs and synthetic polymers, classical polymer models offer a powerful approach for investigating the conformations, dynamics, and phase behavior of IDPs. − In particular, such models have been extensively used to reveal the sequence-dependent conformations of IDPs in solution and in condensates. − …”

Role of Strong Localized vs Weak Distributed Interactions in Disordered Protein Phase Separation

“…14 These prior works suggest that principles from homopolymer theory can be applied with reasonable accuracy when attractive monomers are distributed rather uniformly along the protein sequence. 25 With these considerations, we investigated whether the similarity established at the single-chain level by scaling monomer interactions also leads to similarities in phase behavior for our model sequences having strong localized or weak distributed interactions. We performed direct coexistence simulations of the dilute and dense phases, computed time-averaged concentration profiles (Figure 4), and extracted the coexistence concentrations of the dilute and dense phases (Figure 5).…”

“…Despite these challenges, significant progress has been made in elucidating the sequence determinants of phase separation through experimental and computational studies. − Among these studies, the work on the low-complexity domain (LCD) of hnRNPA1 , and the FUS prion-like domain have shown that the critical temperature T c and the saturation (dilute-phase) concentration c sat were altered when the number of aromatic residues (tyrosine) was varied. In addition, the mutation of arginine to lysine while maintaining the tyrosine content in FUS-like proteins led to a significant increase in c sat , thus implying a reduction in phase-separation propensity. , Further work on the FUS-LCD, using a combination of nuclear Overhauser experiments and all-atom molecular simulations, demonstrated that residues such as serine, glycine, and glutamine also play an important role in the stabilization of liquid-like condensates through interactions with other residues in the sequence. , Studies involving shuffling of charges in LAF-1 RGG and DDX4 have shown that charge patterning, frequently quantified by the sequence charge decoration parameter, influences the phase-separation propensity of a protein, highlighting the key role played by electrostatic interactions.…”

“…Another class of protein models considers the collective contribution of all amino acids in a protein sequence by employing weak interactions, comprised of various interaction modes, distributed along the protein. ,, Transferable coarse-grained models developed by some of us, using amino acid specific contact potentials based on hydrophobicity, are reflective of this consideration and have been used to successfully characterize the phase behavior of reference proteins like FUS-LCD, LAF-1 RGG, α-synuclein, and A1-LCD. ,,− An extension of the model applying an additional temperature-dependent parabolic scaling for the nonbonded interactions was shown to capture experimentally observed upper critical or lower critical solution temperature transitions for a set of thermoresponsive model IDPs …”