Protein–carbohydrate interactions are essential for numerous physiological and pathological processes, including host–pathogen recognition, immune modulation, and microbial adhesion. Despite their biological relevance, the intrinsically low affinity and transient nature of these interactions often hinder their structural and functional characterization using conventional techniques. This doctoral thesis addresses this challenge by exploiting and refining ligand-based NMR spectroscopy, with a particular focus on the Saturation Transfer Difference (STD) NMR technique, to explore weak protein–ligand interactions in systems of biological relevance.
The main contributions include:
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Development of IL-STD NMR, a new methodology that enables the detection of spatial proximity between ligands within binding pockets containing multiple subsites. IL-STD NMR is first validated in a model system and subsequently successfully applied to a biologically relevant system, such as the B subunit of cholera toxin, highlighting its broad applicability.
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Study of High-Mannose recognition by DC-SIGN, involving the development of a combined strategy based on the synthesis of a selectively fluorinated nonamannoside (F-Man9), epitope mapping using 2D ^1H,^19F STD-TOCSYreF build-up curves, and molecular dynamics simulations, demonstrating that F-Man9 can be used as a High-Mannose glycomimetic. This strategy enabled highly refined epitope mapping and provided the first 3D molecular model of the DC-SIGN–Man9 complex.
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Investigation of the inhibition mechanism of the bacterial enzyme NleB1 by YM155, through the development of a novel STD NMR–based strategy termed “Epitope Perturbation by Mutation.” This approach involves site-directed mutagenesis to enable precise mapping of the inhibitor binding site. Complementary docking simulations and molecular dynamics simulations were used to generate a 3D molecular model of the inhibitor–enzyme complex, providing detailed insight into ligand recognition and revealing the molecular basis of inhibition.