A synergy between site-specific and transient interactions drives the phase separation of a disordered, low-complexity domain

Abstract

TAR DNA-binding protein 43 (TDP-43) is involved in key processes in RNA metabolism and is frequently implicated in many neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. The prion-like, disordered C-terminal domain (CTD) of TDP-43 is aggregation-prone, can undergo liquid-liquid phase separation (LLPS) in isolation, and is critical for phase separation (PS) of the full-length protein under physiological conditions. While a short conserved helical region (CR, spanning residues 319-341) promotes oligomerization and is essential for LLPS, aromatic residues in the flanking disordered regions (QN-rich, IDR1/2) are also found to play a critical role in PS and aggregation. Compared with other phase-separating proteins, TDP-43 CTD has a notably distinct sequence composition including many aliphatic residues such as methionine and leucine. Aliphatic residues were previously suggested to modulate the apparent viscosity of the resulting phases, but their direct contribution toward CTD phase separation has been relatively ignored. Using multiscale simulations coupled with in vitro saturation concentration (c sat ) measurements, we identified the importance of aromatic residues while also suggesting an essential role for aliphatic methionine residues in promoting single-chain compaction and LLPS. Surprisingly, NMR experiments showed that transient interactions involving phenylalanine and methionine residues in the disordered flanking regions can directly enhance site-specific, CR-mediated intermolecular association. Overall, our work highlights an underappreciated mode of biomolecular recognition, wherein both transient and site-specific hydrophobic interactions act synergistically to drive the oligomerization and phase separation of a disordered, low-complexity domain.

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

“…Hence, the established molecular grammar for phase separation of prion-like domains could be expanded to include glutamine at the very least. Indeed, we find that glutamine enhances phase separation much more than alanine, consistent with our previous results showing that methionine also contributes more than alanine to TDP-43 phase separation 30 . Alternatively, given the role of many different residue pair contacts in phase separation, one could adopt a non-binary inclusive classification where most (if not all) residues can contribute toward molecular driving forces even if primarily via forming stabilizing contacts with other residue types, depending on the context and other environmental factors such as temperature, salt, and pH [80][81][82] .…”

A complex network of interdomain interactions underlies the conformational ensemble of monomeric TDP‐43 and modulates its phase behavior

“…Both NTD and CTD participated in homodomain interactions which is consistent with their propensity to undergo selfoligomerization. In agreement with previous studies, both CR and IDR regions of the CTD contribute extensively toward favorable interactions in the condensed phase (Conicella et al, 2020;Hallegger et al, 2021;Mohanty et al, 2023;Schmidt et al, 2019). Interestingly, RRM1/2 and disordered linkers-L1/L2 also participated extensively in contact formation (Figure 3b).…”

Toward Accurate Simulation of Coupling between Protein Secondary Structure and Phase Separation

“…Examination of the probability map of residue–residue contacts in the condensate of the WT TDP-43 CR, shown in Figure A, reveals that three aromatic residues, F313, F316, and W334, play dominant roles in mediating intermolecular interactions. Previous in vitro and in vivo experiments also suggest that aromatic residues play critical roles in the phase separation of TDP-43. − It is noteworthy that the helical regions do not appear to be a major contributor to intermolecular contacts in the condensed phase. Inspection of the distribution of partial helices within the condensates reveals that helical regions spread around uniformly in the condensed phase, and there is no apparent tendency for preferential helix–helix contacts ( e.g.…”