The VP8* subunit of rotavirus spike
protein VP4 contains a
sialic acid (Sia)-binding domain important for host cell attachment and
infection. In this study, the binding
epitope of the
N-acetylneuraminic acid (Neu5Ac) derivatives has been characterized by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. From this STD NMR data, it is proposed that the VP8* core recognizes an identical binding
epitope in both
methyl alpha-D-N-acetylneuraminide (Neu5Acalpha2Me) and the
disaccharide methyl S-(alpha-D-N-acetylneuraminosyl)-(2-->6)-6-thio-beta-D-
galactopyranoside (Neu5Ac-alpha(2,6)-S-Galbeta1Me). In the VP8*-
disaccharide complex, the Neu5Ac moiety contributes to the majority of interaction with the
protein, whereas the
galactose moiety is
solvent-exposed. Molecular dynamics calculations of the VP8*-
disaccharide complex indicated that the
galactose moiety is unable to adopt a conformation that is in close proximity to the
protein surface. STD NMR experiments with
methyl 9-O-acetyl-alpha-D-N-acetylneuraminide (
Neu5,9Ac(2)alpha2Me) in complex with rhesus rotavirus (RRV) VP8* revealed that both the N-
acetamide and 9-O-acetate moieties are in close proximity to the Sia-binding domain, with the N-
acetamide's methyl group being saturated to a larger extent, indicating a closer association with the
protein. RRV VP8* does not appear to significantly recognize the unsaturated Neu5Ac derivative [2-deoxy-2,3-didehydro-D-
N-acetylneuraminic acid (
Neu5Ac2en)]. Molecular modeling of the protein-Neu5Ac2en complex indicates that key interactions between the
protein and the unsaturated Neu5Ac derivative when compared with Neu5Acalpha2Me would not be sustained. Neu5Acalpha2Me, Neu5Ac-alpha(2,6)-S-Galbeta1Me,
Neu5,9Ac(2)alpha2Me, and
Neu5Ac2en inhibited
rotavirus infection of MA104 cells by 61%, 35%, 30%, and 0%, respectively,
at 10 mM concentration. NMR spectroscopic, molecular modeling, and infectivity inhibition results are in excellent agreement and provide valuable information for the design of inhibitors of
rotavirus infection.