The transactive response (TAR) DNA/RNA‐binding protein 43 (TDP‐43) can self‐assemble into both functional stress granules via liquid–liquid phase separation (LLPS) and pathogenic amyloid fibrillary aggregates that are closely linked to amyotrophic lateral sclerosis. Previous experimental studies reported that the low complexity domain (LCD) of TDP‐43 plays an essential role in the LLPS and aggregation of the full‐length protein, and it alone can also undergo LLPS to form liquid droplets mainly via intermolecular interactions in the 321–340 region. And the ALS‐associated M337V mutation impairs LCD's LLPS and facilitates liquid–solid phase transition. However, the underlying atomistic mechanism is not well understood. Herein, as a first step to understand the M337V‐caused LLPS disruption of TDP‐43 LCD mediated by the 321–340 region and the fibrillization enhancement, we investigated the conformational properties of monomer/dimer of TDP‐43321–340 peptide and its M337V mutant by performing extensive all‐atom explicit‐solvent replica exchange molecular dynamic simulations. Our simulations demonstrate that M337V mutation alters the residue regions with high helix/β‐structure propensities and thus affects the conformational ensembles of both monomer and dimer. M337V mutation inhibits helix formation in the N‐terminal Ala‐rich region and the C‐terminal mutation site region, while facilitating their long β‐sheet formation, albeit with a minor impact on the average probability of both helix structure and β‐structure. Further analysis of dimer system shows that M337V mutation disrupts inter‐molecular helix–helix interactions and W334‐W334 π‐π stacking interactions which were reported to be important for the LLPS of TDP‐43 LCD, whereas enhances the overall peptide residue‐residue interactions and weakens peptide‐water interactions, which is conducive to peptide fibrillization. This study provides mechanistic insights into the M337V‐mutation‐induced impairment of phase separation and facilitation of fibril formation of TDP‐43 LCD.
Liquid-liquid phase separation (LLPS) of proteins mediates the assembly of biomolecular condensates involved in physiological and pathological processes. Identifying the minimalistic building blocks and the sequence determinant of protein phase separation is of urgent importance but remains challenging due to the enormous sequence space and difficulties of existing methodologies in characterizing the phase behavior of ultrashort peptides. Here we demonstrate computational tools to efficiently quantify the microscopic fluidity and density of liquid-condensates/solid-aggregates and the temperature-dependent phase diagram of peptides. Utilizing our approaches, we comprehensively predict the LLPS abilities of all 400 dipeptide combinations of coded amino acids based on 492 micro-second molecular dynamics simulations, and observe the occurrences of spontaneous LLPS. We identify 54 dipeptides that form solid-like aggregates and three categories of dipeptides with high LLPS propensity. Our predictions are validated by turbidity assays and differential interference contrast (DIC) microscopy on four representative dipeptides (WW, QW, GF, and VI). Phase coexistence diagrams are constructed to explore the temperature dependence of LLPS. Our results reveal that aromatic moieties are crucial for a dipeptide to undergo LLPS, and hydrophobic and polar components are indispensable. We demonstrate for the first time that dipeptides, minimal but complete, possess multivalent interactions sufficient for LLPS, suggesting that LLPS is a general property of peptides/proteins, independent of their sequence length. This study provides a computational and experimental approach to the prediction and characterization of the phase behavior of minimalistic peptides, and will be helpful for understanding the sequence-dependence and molecular mechanism of protein phase separation.
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