Interaction studies by NMR on the multivalent interaction between chondroitin sulfate E derivatives and the langerin...

In this paper, we present the NMR analysis of multivalent compounds displaying chondroitin sulfate E (CS-E) disaccharide ligands and their interaction with langerin. The disaccharides correspond to the two alternative sequences of CS-E: GlcA-GalNAc and GalNAc-GlcA. Firstly, we studied the conformation of the two corresponding series of glycodendrimers free in solution and in the presence of langerin. The NMR structures of the free compounds are compatible with the expected ones. Both sequences exhibit very similar conformations with a rigid moiety, the disaccharides, and a flexible region corresponding to the rest of the molecule. A key aspect of this work is the detailed analysis of how different compounds interact with distinct regions of the langerin receptor as a function of the spatial distribution of the same binding epitope. This allows us to gain unique insights into the receptor's binding behavior and the specific interactions mediated by different ligand multivalency. We conducted transferred NOESY experiments in the presence of langerin, concluding that the conformations of the bound disaccharides were the same as the free ones. In addition, we performed STD-NMR experiments and analyzed the binding epitope, demonstrating that the monovalent compound with GlcA at the non-reducing end can interact with langerin through the Ca²⁺ cation, while the reverse sequence does not. The corresponding trimers 2 and 6 interact mainly via the central aromatic core, independent of the disaccharide sequence. In the case of tetramer 3, the interaction takes place mainly by the GalNAc proton in position 4 and in the hexamer, significant spin diffusion prevents epitope analysis, although the interaction with langerin is clearly observed. The STD-NMR experiments in the absence of Ca²⁺ showed a lack of binding for both monovalent compounds 1 and 5. In contrast, in the case of multivalent compounds 2, 3 and 6, STD peak characteristics of binding were found with similar pattern epitope maps to those obtained in the presence of Ca²⁺. We also performed DOSY experiments for the first series of GlcA-GalNAc compounds individually, in the presence and absence of langerin, and for the mixture of all the compounds in the same NMR tube. Finally, we performed MD simulations for the monovalent and trivalent compounds, corroborating the NMR analysis for the compounds in the absence of langerin.