Evolution of intrinsically disordered regions in vertebrate galectins for phase separation.

Intrinsically disordered regions (IDRs) are widespread in proteins, yet their evolutionary paths remain poorly understood. Using galectin, a universal carbohydrate-binding protein, we investigated how IDRs evolved and acquired their biological roles in vertebrates. Through extensive proteome-wide sequence analyses, we found that vertebrate galectin IDRs share overall amino acid compositions but differ significantly in their aromatic residue types. Using nuclear magnetic resonance (NMR) spectroscopy and lipopolysaccharide micelle assays, we demonstrated that despite these differences, IDRs from various vertebrate galectins independently converged toward a similar function: mediating agglutination via phase separation. Our data suggest that the specific types of aromatic residues within these IDRs were established early in evolution and underwent independent expansions among different vertebrate lineages. Additionally, we identified a conserved short N-terminal motif critical for promoting galectin self-association, which likely served as an incipient sequence for subsequent IDR evolution. Contrary to previous peptide studies emphasizing aromatic residue specificity, our findings highlight the evolutionary preference for increasing motif repetition over residue-type optimization to achieve functional fitness.