Structural Basis for Human Milk Oligosaccharides Recognition by Evolutionarily Dominant Norovirus Genogroup II Genotypes

Human noroviruses are a major cause of acute gastroenteritis. Human milk oligosaccharides (HMOs) contain glycan motifs similar to histo-blood group antigens and may act as soluble decoys that reduce viral attachment. However, the molecular basis of HMO recognition by the P domain of the major capsid protein VP1 of different norovirus genotypes is not fully understood. Here, we combined ligand-based and protein-based NMR experiments with X-ray crystallography to study HMO binding to the P domain of the latent GII.2, dominant GII.4 and emerging GII.17 noroviruses genotypes. Screening of 12 HMOs by STD NMR showed binding of all fucosylated HMOs to GII.4 and GII.17 and none for non-fucosylated HMOs. In contrast, GII.2 showed little or no detectable binding under the same conditions. Seven crystal structures of GII.4 or GII.17 with complexed HMOs supported fucose anchoring via five conserved residues. The remaining sugar moieties formed genotype-specific secondary interactions with an extended, shallow and solvent exposed binding surface whose shape is dictated by divergent surface loops. GII.4 recognition is mainly driven by hydrogen bonds whereas GII.17 relied more strongly on water-mediated interactions. Chemical shift perturbation from NMR titrations (CSP NMR) of GII.4 corroborated the fucose interactions and gave dissociation constants of 20-33 mM, consistent with weak and rapidly exchanging interactions.