| Sumario: | 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 intrinsic low affinity and transient nature of these interactions often hinders their structural and functional characterization using conventional techniques. This doctoral thesis addresses this challenge by leveraging and advancing ligand-observed NMR spectroscopy, with a particular focus on Saturation Transfer Difference (STD) NMR, to explore weak protein-ligand interaction in biologically significant systems. The work in this thesis dissertation presents several methodological innovations aimed at extracting deeper structural information from weak protein-ligand complexes. In Chapter 3, a new approach called Inter-Ligand STD NMR (IL-STD NMR) is developed and validated. This technique allows the detection of inter-ligand spatial proximity within multi-subsite protein binding pockets. IL-STD NMR is first validated on a model system and, subsequently, successfully applied to a biologically relevant system such as the cholera toxin B subunit (CTB), highlighting its broad applicability. In Chapter 4, this thesis also explores the use of 2D 1H,19F STD-TOCSYreF experiments and implements for the first time the initial slope analysis of the full 2D 1H,19F STD-TOCSYreF build-up curves to investigate the recognition of high-mannose oligosaccharide by DC-SIGN, a key lectin in pathogen-host interactions. A combined strategy based on the synthesis of a selectively fluorinated nonamannoside (F-Man9), epitope mapping by 2D 1H,19F STD-TOCSYreF build-up curves, and Molecular Dynamics (MD) simulations, demonstrates that F-Man9 can be employed as a glycomimetic of high-mannose (Man9), leads to a highly refined epitope mapping, and yields the first 3D molecular model of the DC-SIGN-Man9 complex. Finally, Chapter 5 explores the inhibition mechanism of the pathogenic E. coli enzyme NleB1 by YM155, a recently identified potent small-molecule inhibitor of the NleB/SseK family of arginine glycosyltransferases (Arg-GTs), which are bacterial effectors involved in virulence. To investigate the process of molecular recognition of YM155 by NleB1, a novel STD NMR-based strategy, Epitope Perturbation by Mutation, is developed. This approach involves site-directed mutagenesis to enable precise mapping of the ligand binding site. Complementary docking calculations and molecular dynamics simulations are used to generate a 3D molecular model of the inhibitor-enzyme complex, providing detailed insights into ligand recognition and revealing the molecular basis of inhibition. The results indicate that the inhibition mechanism of YM155 is based on the induction of a conformational change in the bound state of the side chain of the acceptor arginine residue, rendering a geometry unfavourable for the transfer of GlcNAc from the donor substrate.
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