Molecular recognition of the Thomsen-Friedenreich antigen-threonine conjugate by adhesion/growth regulatory galectin-3: nuclear magnetic resonance studies and molecular dynamics simulations.

Nuclear magnetic resonance (NMR) spectroscopy and molecular modeling methods have been strategically combined to elucidate the molecular recognition features of the binding of threonine O-linked Thomsen-Friedenreich (TF) antigen to chimera-type avian galectin-3 (CG-3). Saturation transfer difference (STD) NMR experiments revealed the highest intensities for the H4 protons of both the beta-D-Galp and a-D-GalpNAc moieties, with 100 and 71% of relative STD, respectively. The methyl protons of the threonine residue exhibited a small STD effect, <15%, indicating that the interaction of the amino acid with the protein is rather transient. Two-dimensional transferred nuclear Overhauser effect spectroscopy NMR experiments and molecular modeling suggested some differences in conformer populations between the free and bound states. A dynamic binding mode for the TF antigen-CG-3 complex consisting of two poses has been deduced. In one pose, interactions were formed between the terminal threonine residue and the receptor. In the second pose, intermolecular interactions involved the internal GalpNAc. The difference in the trend of some shifts in the heteronuclear single-quantum coherence titration spectra indicates some disparities in the binding interactions of CG-3 with lactose and TF antigen. The results obtained from this model of the avian orthologue of human galectin-3 will allow detailed interspecies comparison to give sequence deviations in phylogeny a structural and functional meaning. Moreover, the results indicate that I:he peptide scaffold presenting TF antigen could be relevant for binding and thus provides a possible route for the design of galectin-3 inhibitors with improved affinity and selectivity.