Abstract The recognition of carbohydrates by lectins play key roles in diverse cellular processes such as cellular adhesion, proliferation and apoptosis which makes it a promising therapeutic target against cancers. One of the most functionally active lectins, galectin-3 is distinctively known for its specific binding affinity towards β -galactoside. Despite the prevalence of high-resolution crystallographic structures, the mechanistic basis and the molecular determinants of the sugar recognition process by galectin-3 are currently elusive. Here we address this question by capturing the complete dynamical binding process of human galectin-3 with its native ligand N-acetyllactosamine (LacNAc) and one of its synthetic derivatives by unbiased Molecular Dynamics simulation. In our simulations, both the natural ligand LacNAc and its synthetic derivative, initially solvated in water, diffuse around the protein and eventually recognise the designated binding site at the S-side of galectin-3, in crystallographic precision and identifies key metastable intermediate ligand-states around the galectin on their course to eventual binding. The simulations highlight that the origin of the experimentally observed multi-fold efficacy of synthetically designed ligand-derivative over its native natural ligand LacNAc lies in the derivative’s relatively longer residence time in the bound pocket. A kinetic analysis demonstrates that the LacNAc-derivative would be more resilient compared to the parent ligand against unbinding from the protein binding site. In particular, the analysis identifies that interactions of the binding pocket residues Trp181, Arg144 and Arg162 with the tetrafuorophenyl ring of the derivative as the key determinant for the synthetic ligand to latch into the pocket. Graphical Abstract

Molecular Determinants of Ligand Residence in Galectin