Protein structural transitions critically transform the network connectivity and viscoelasticity of RNA-binding protein condensates but RNA can prevent it

Abstract

Biomolecular condensates, some of which are liquid-like during health, can age over time becoming gel-like pathological systems. Ageing of RNA-binding protein condensates can emerge from the progressive accumulation of inter-protein β-sheets. To bridge microscopic understanding of such time-dependent transformation with the modulation of FUS and hnRNPA1 condensate viscoelasticity, we develop a multiscale simulation approach. Our method integrates atomistic simulations with sequence-dependent coarse-grained modelling of condensates that age over time due to accumulation of inter-protein β-sheets. We reveal that ageing notably increases condensate viscosity but does not transform the phase diagrams. Strikingly, the network of molecular connections within condensates is drastically altered during ageing and culminates in gelation when the network of strong inter-protein β-sheets fully percolates. High concentrations of RNA decelerate the accumulation of inter-protein β-sheets, abrogating the effects of ageing. Our study uncovers molecular and kinetic factors explaining condensate ageing, and suggests a potential mechanism to slow ageing down.

Location and concentration of aromatic-rich segments dictates the percolating inter-molecular network and viscoelastic properties of ageing condensates

“…Furthermore, LARKS abundance is also critical for controlling droplet densification upon maturation, where the relative increase in density is 2.5 times higher from 1-LARKS to 3-LARKS low-complexity domains. These results are consistent with previous in silico observations for FUS-LCD and A1-LCD-hnRNPA1 aged condensates, where the higher concentration of LARKS within the FUS-LCD sequence led to higher densification upon maturation compared to A1-LCD-hnRNPA1 droplets 31 . Recently, FUS-LCD ageing-driven densification through β -sheet aggregation has been confirmed 11,65 .…”

“…Furthermore, as reported in Fig. 1(b), the relatively high interaction strengths among structured LARKS that we obtain here for TDP-43 are also consistent with all-atom PMF calculations for NUP-98 55 , hnRNPA1 31 and FUS 32 , previously reported by us. For all these proteins, a dramatic increase in binding energy from the disordered state (where interaction energies typically correspond to 3-8 k B T per peptide, suggesting that thermal fluctuations can easily break them) is observed upon the formation of the canonical β -sheet stacking (~30-45 k B T of interaction per peptide).…”

“…(b) Overview of the structured vs . disordered LARKS binding free energies found in the low-complexity domains of FUS 32 , NUP-98 55 , hnRNPA1 31 and TDP-43 (this work). Snapshots of the atomistic LARKS sequences, included in the inset and specified below, are coloured by amino acid identity.…”

“…1(c)). Then, through our dynamic algorithm 31,32 , we approximate the non-equilibrium process of condensate ageing by considering the atomistic implications (i.e., non-conservative strengthening of inter-protein binding, local protein rigidification, and changes in the inter-molecular organization; Fig. 1(a)) of the gradual and irreversible accumulation of inter-protein β -sheet structures in a time-dependent manner, and as a function of the local protein density.…”

“…The formation of inter-protein kinked β -sheets has been postulated as an intrinsic mechanism that can explain how condensates may undergo ageing without the need to invoke chemical changes in the system or in the environmental conditions 28 . Low-complexity domains (LCDs) with high aromatic content, which are important contributors to the multivalency of many naturally occurring proteins 4,29,30 , can exhibit local disorder-to-order structural transitions inside condensates, forming inter-protein β -sheets 14,28,31,32 and, subsequently, amyloid fibrils 20,33,34 . The accumulation of inter-protein β -sheet structures formed by low-complexity aromatic-rich kinked segments (LARKS) produces concomitant enhancement of inter-protein interactions and can drive a gradual phase transition from functional liquid-like states into less dynamic reversible hydrogels or even irreversible solid-like states 7,10,35,36 .…”