Phosphorylation sites are evolutionary checkpoints against liquid-solid transition in protein condensates

Assemblies of multivalent RNA-binding protein FUS can exist in the functional liquid-like state as well as less dynamic and potentially toxic amyloid- and hydrogel-like states. How could then cells form liquid-like condensates while avoiding their transformation to amyloids? Here we show how post-translational phosphorylation can provide a “handle” that prevents liquid-solid transition of intracellular condensates containing FUS. Using residue-specific coarse-grained simulations, for 85 different mammalian FUS sequences, we show how the number of phosphorylation sites and their spatial arrangement affect intracluster dynamics preventing conversion to amyloids. All atom simulations further confirm that phosphorylation can effectively reduce the β-sheet propensity in amyloid-prone fragments of FUS. A detailed evolutionary analysis shows that mammalian FUS PLDs are enriched in amyloid-prone stretches compared to control neutrally evolved sequences suggesting that mammalian FUS proteins evolved to self-assemble. However, in stark contrast to proteins that do not phase-separate for their function, mammalian sequences have phosphosites in close proximity to these amyloid-prone regions. These results suggest that evolution uses amyloid-prone sequences in prion-like domains to enhance phase-separation of condensate proteins while enriching phosphorylation sites in close proximity to safe-guard against liquid-solid transitions. Significance Statement Intrinsically disordered regions and prion-like domains are widely observed in proteins that get enriched in membrane-less organelles (MLOs). Mammalian Fused in Sarcoma (FUS) sequences are significantly enriched in amyloid-prone sequences suggesting that they have evolved to self-assemble. While the amyloid-prone stretches promote self-assembly of these proteins at lower threshold concentrations, these assemblies are vulnerable to aberrant liquid-solid phase transitions. Molecular simulations and bioinformatics analyses show that evolution overcomes this challenge by placing phosphosites specifically close to amyloid-prone stretches. Introduction of negatively charged residues at phosphosite locations results in fewer amyloid-prone contacts and thereby lower beta-sheet propensity. Phosphorylation can thus allow cells to utilize amyloid-prone stretches to promote biogenesis of MLOs while protecting against liquid-solid transitions. ### Competing Interest Statement The authors have declared no competing interest.