The mutational landscape of a Prion-like domain

Specific insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, cytoplasmic aggregates of the RNA-binding protein TDP-43 are observed in 97% of cases of Amyotrophic Lateral Sclerosis (ALS). However, whether the protein aggregates themselves or other forms of the proteins are toxic to cells is still a very open question for many of these diseases. Here we address this question for TDP-43 by systematically mutating the protein and quantifying the effects on cellular toxicity.  In a dataset of >50,000 mutations in the intrinsically disordered prion-like domain (PRD), changes in hydrophobicity and aggregation potential are highly predictive of changes in toxicity. Surprisingly, however, increased hydrophobicity and cytoplasmic aggregation reduce toxicity. Mutations have their strongest effects in a central region of the PRD, with variants that increase toxicity promoting the formation of more dynamic liquid-like condensates. Moreover, the genetic interactions in double mutants indicate that specific secondary structures form in this region and have detectable effects on aggregation and toxicity in vivo. Our results demonstrate that deep mutagenesis is a powerful approach for probing the sequence-function relationships of intrinsically disordered proteins, as well as their in vivo structural conformations.  Moreover, they reveal that aggregation of TDP-43 is not toxic but actually protects cells, most likely by titrating the protein away from a toxic liquid-like phase. Systematic measurment of the toxic effects of 50.000 mutations in the region 290-373 of TDP-43 Input 5-8 are biological replicates for library TDP aa 290-331; Input 1-4 are biological replicates for library TDP aa 332-373. Each input corresponds to DNA extracted from cells growing after transformation and without expression of TDP-43. Outputs named with the same number are technical replicates and they originate from the same input. Each output corresponds to DNA extracted from cells growing after having expressed TDP-43 for roughly 6 generations.