A Few Charged Residues in Galectin‐3′s Folded and Disordered Regions Regulate Phase Separation

Abstract Proteins with intrinsically disordered regions (IDRs) often undergo phase separation to control their functions spatiotemporally. Changing the pH alters the protonation levels of charged sidechains, which in turn affects the attractive or repulsive force for phase separation. In a cell, the rupture of membrane‐bound compartments, such as lysosomes, creates an abrupt change in pH. However, how proteins’ phase separation reacts to different pH environments remains largely unexplored. Here, using extensive mutagenesis, NMR spectroscopy, and biophysical techniques, it is shown that the assembly of galectin‐3, a widely studied lysosomal damage marker, is driven by cation‐π interactions between positively charged residues in its folded domain with aromatic residues in the IDR in addition to π–π interaction between IDRs. It is also found that the sole two negatively charged residues in its IDR sense pH changes for tuning the condensation tendency. Also, these two residues may prevent this prion‐like IDR domain from forming rapid and extensive aggregates. These results demonstrate how cation‐π, π–π, and electrostatic interactions can regulate protein condensation between disordered and structured domains and highlight the importance of sparse negatively charged residues in prion‐like IDRs.