title: STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core.
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 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.
authors.affiliation: Institute for Glycomics, Griffith University, Gold Coast Campus, PMB 50 Gold Coast Mail Centre, Queensland 9726, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Haselhorst
authors.forename: Helen
authors.initials: H
authors.lastname: Blanchard
authors.forename: Martin
authors.initials: M
authors.lastname: Frank
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Kraschnefski
authors.forename: Milton J
authors.initials: MJ
authors.lastname: Kiefel
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Szyczew
authors.forename: Jeffery C
authors.initials: JC
authors.lastname: Dyason
authors.forename: Fiona
authors.initials: F
authors.lastname: Fleming
authors.forename: Gavan
authors.initials: G
authors.lastname: Holloway
authors.forename: Barbara S
authors.initials: BS
authors.lastname: Coulson
authors.forename: Mark
authors.initials: M
authors.lastname: von Itzstein
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008954:Models, Biological; D008958:Models, Molecular; D008968:Molecular Conformation; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D012401:Rotavirus / Q000187:drug effects; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D004187:Disaccharides; D016601:RNA-Binding Proteins; D012794:Sialic Acids; D017361:Viral Nonstructural Proteins; C518197:methyl 9-O-acetyl-N-acetylneuraminide; C518195:methyl N-acetylneuraminide; C518196:methyl S-(N-acetylneuraminosyl)-(2-6)-6-thiogalactopyranoside; C079234:NS35 protein, rotavirus; C036137:2-deoxy-2,3-dehydro-N-acetylneuraminic acid; C025676:9-O-acetyl-N-acetylneuraminic acid; D019158:N-Acetylneuraminic Acid