The mutational landscape of a prion-like domain

Insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, aggregates of TDP-43 occur in nearly all cases of amyotrophic lateral sclerosis (ALS). However, whether aggregates cause cellular toxicity is still not clear, even in simpler cellular systems. We reasoned that deep mutagenesis might be a powerful approach to disentangle the relationship between aggregation and toxicity. We generated >50,000 mutations in the prion-like domain (PRD) of TDP-43 and quantified their toxicity in yeast cells. Surprisingly, mutations that increase hydrophobicity and aggregation strongly decrease toxicity. In contrast, toxic variants promote the formation of dynamic liquid-like condensates. Mutations have their strongest effects in a hotspot that genetic interactions reveal to be structured in vivo, illustrating how mutagenesis can probe the in vivo structures of unstructured proteins. Our results show that aggregation of TDP-43 is not harmful but protects cells, most likely by titrating the protein away from a toxic liquid-like phase.Work in B.L.’s lab was supported by a European Research Council (ERC) Consolidator grant (616434), the Spanish Ministry of Economy and Competitiveness (BFU2017-89488-P), the AXA Research Fund, the Bettencourt Schueller Foundation, and Agencia de Gestio d’Ajuts Universitaris i de Recerca (AGAUR, SGR-831) G.G.T.’s lab was supported by the European Research Council (RIBOMYLOME_309545) and the Spanish Ministry of Economy and Competitiveness (BFU2014-55054-P and BFU2017-86970-P). We acknowledge support from the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, the EMBL Partnership, and the CERCA Program/Generalitat de Catalunya. We thank Pablo Baeza Centurión, Xavier Salvatella, Alexandros Armaos and Benjamin Lang for discussion and assistance and the Eisenberg lab for help with the ZipperDB analysis