The intrinsically disordered N-terminal domain of galectin-3 dynamically mediates multisite self-association of the protein through fuzzy interactions

Galectins are a family of lectins that bind β-galactosides through their conserved carbohydrate recognition domain (CRD) and can induce aggregation with glycoproteins or glycolipids on the cell surface and thereby regulate cell activation, migration, adhesion, and signaling. Galectin-3 has an intrinsically disordered N-terminal domain and a canonical CRD. Unlike the Other 14 known galectins in mammalian cells, which have dimeric or tandem-repeated CRDs enabling multivalency for various functions, Galectin-3 is monomeric, and its functional multivalency therefore is somewhat of a mystery. Here, we used NMR spectroscopy, mutagenesis, small-angle X-ray scattering, and computational modeling to study the self-association-related multivalency of Galectin-3 at the residue-specific level. We show that the disordered N-terminal domain (residues ∼20-100) interacts with itself and with a part of the CRD not involved in carbohydrate recognition (β-strands 7-9; residues ∼200-220), forming a fuzzy complex via inter- and intramolecular interactions, mainly through hydrophobicity. These fuzzy interactions are characteristic of intrinsically disordered proteins to achieve liquid-liquid phase separation, and we demonstrated that Galectin-3 can also undergo liquid-liquid phase separation. We propose that Galectin-3 may achieve multivalency through this multisite self-association mechanism facilitated by fuzzy interactions.

galectin; intrinsically disordered protein; nuclear magnetic resonance (NMR); protein dynamic; protein folding; protein self-assembly.