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STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core

Journal Article


Abstract


  • 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 α-D-N-acetylneuraminide (Neu5Acα2Me) and the disaccharide methyl S- (α-D-N -acetylneuraminosyl)-(2→6)-6-thio-β-D-galactopyranoside (Neu5Ac-α(2,6)-S-Galβ1Me). 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-α-D-N-acetylneuraminide (Neu5, 9Ac2α2Me) 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 Neu5Acα2Me would not be sustained. Neu5Acα2Me, Neu5Ac-α(2,6)-S-Galβ1Me, Neu5,9Ac2α2Me, 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. © 2007 Oxford University Press.

UOW Authors


  •   Blanchard, Helen (external author)

Publication Date


  • 2007

Citation


  • Haselhorst, T., Blanchard, H., Frank, M., Kraschnefski, M. J., Kiefel, M. J., Szyczew, A. J., . . . Von Itzstein, M. (2007). STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core. Glycobiology, 17(1), 68-81. doi:10.1093/glycob/cwl051

Scopus Eid


  • 2-s2.0-33845568286

Start Page


  • 68

End Page


  • 81

Volume


  • 17

Issue


  • 1

Abstract


  • 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 α-D-N-acetylneuraminide (Neu5Acα2Me) and the disaccharide methyl S- (α-D-N -acetylneuraminosyl)-(2→6)-6-thio-β-D-galactopyranoside (Neu5Ac-α(2,6)-S-Galβ1Me). 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-α-D-N-acetylneuraminide (Neu5, 9Ac2α2Me) 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 Neu5Acα2Me would not be sustained. Neu5Acα2Me, Neu5Ac-α(2,6)-S-Galβ1Me, Neu5,9Ac2α2Me, 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. © 2007 Oxford University Press.

UOW Authors


  •   Blanchard, Helen (external author)

Publication Date


  • 2007

Citation


  • Haselhorst, T., Blanchard, H., Frank, M., Kraschnefski, M. J., Kiefel, M. J., Szyczew, A. J., . . . Von Itzstein, M. (2007). STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core. Glycobiology, 17(1), 68-81. doi:10.1093/glycob/cwl051

Scopus Eid


  • 2-s2.0-33845568286

Start Page


  • 68

End Page


  • 81

Volume


  • 17

Issue


  • 1