Carbohydrates used in structural studies by NMR are rarely ¹³C-labeled, preventing exploitation of the ¹³C spectral dispersion in 3D or higher order NMR experiments, which would alleviate severe resonance overlap in their ¹H NMR spectra. This is in part associated with the lack of easily accessible isotope-labeled sugar molecules. We previously reported a number of attractive advantages of using ¹³C-labeled carbohydrates to characterize the interactions in protein complexes, comprising ¹⁵N-labeled protein. As model system we used the complex of the ¹³C-labeled Manα(1–2)Manα(1–2)ManαOMe trisaccharide, bound to cyanovirin-N (CV-N). The carbohydrate-protein binding interface was characterized by tailored isotope-filtered NOESY experiments  and carbohydrate hydroxyl protons that form hydrogen bonds with the protein were identified and their orientations were determined .
In this presentation, we report results about CV-N recognition by Manα(1–2)ManαOMe and Manα(1–2)Manα(1–6)ManαOMe. These two mannosides bind preferentially to the domain B binding site of CV-N, in contrast to Manα(1–2)Manα(1–2)ManαOMe, which binds preferentially to the domain A binding site [1, 2].
Intra- and intermolecular NOEs between carbohydrate ring and hydroxyl protons and protein amide protons were investigated. Five carbohydrate hydroxyl protons were identified (Fig. 1) and for four of them, the dihedral angles were determined. Analysis of the current results in light of previous NMR and crystal structures of the same complexes revealed important differences and similarities. These will be highlighted in the presentation. Overall, our results emphasize the general applicability of our novel approach for characterizing hydrogen bonding in carbohydrate-protein interactions.
- G. Nestor, T. Anderson, S. Oscarson, A. M. G. Gronenborn, J. Am. Chem. Soc. 2017, 139, 6210-6216.
- G. Nestor, T. Anderson, S. Oscarson, A. M. G. Gronenborn, J. Am. Chem. Soc. 2018, 140, 339-345.