Protein language model identifies disordered, conserved motifs implicated in phase separation.

Intrinsically disordered regions (IDRs) play a critical role in phase separation and are essential for the formation of membraneless organelles (MLOs). Mutations within IDRs can disrupt their multivalent interaction networks, altering phase behavior and contributing to various diseases. Therefore, examining the evolutionary constraints of IDRs provides valuable insights into the relationship between protein sequences and phase separation. In this study, we utilized the ESM2 protein language model to map the residue-level mutational tolerance landscapes of IDRs. Our findings reveal that IDRs, particularly those actively participating in phase separation, contain conserved amino acids. This conservation is evident through mutational constraints predicted by ESM2 and supported by direct analyses of multiple sequence alignments. These conserved, disordered amino acids include residues traditionally identified as 'stickers' as well as 'spacers' and frequently form continuous sequence motifs. The strong conservation, combined with their potential role in phase separation, suggests that these motifs may act as functional units under evolutionary selection to support stable MLO formation. Our findings underscore the insights into phase separation's molecular grammar made possible through evolutionary analysis enabled by protein language models.