Glycobiology Advance Access originally published online on September 14, 2006
Glycobiology 2007 17(1):68-81; doi:10.1093/glycob/cwl051
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STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core
2 Institute for Glycomics, Griffith University, Gold Coast Campus, PMB 50 Gold Coast Mail Centre, Queensland 9726, Australia
3 Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010, Australia
1 To whom correspondence should be addressed; Tel: +61 755527016; Fax: +61 755528098; e-mail: m.vonitzstein{at}griffith.edu.au
Received on July 17, 2006; revised on September 12, 2006; accepted on September 13, 2006
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 
-D-N-acetylneuraminide (Neu5Ac2Me) and the disaccharide methyl S-(-D-N-acetylneuraminosyl)-(2
6)-6-thio-ß-D-galactopyranoside (Neu5Ac-(2,6)-S-Galß1Me). 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--D-N-acetylneuraminide (Neu5,9Ac22Me) 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 Neu5Ac2Me would not be sustained. Neu5Ac2Me, Neu5Ac-(2,6)-S-Galß1Me, Neu5,9Ac22Me, 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.
Key words: STD NMR spectroscopy / molecular modelling / rotavirus / VP8* core / lectin
* These authors contributed equally to this work.
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