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

Thomas Haselhorst,Martin Frank,Alex J Szyczew,Mark J Kraschnefski,Gavan Holloway,Barbara S Coulson,Jeffery C Dyason,Milton J Kiefel,Mark Von Itzstein,Helen Blanchard,Fiona Fleming

doi:10.1093/glycob/cwl051

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.

Highlights

Rotaviruses are members of the Reoviridae family and are recognized as the single most important cause of severe gastroenteritis in the infants of a wide range of mammals including humans (Estes 2001)
In order to provide further insight into the nature of the interaction between rhesus rotavirus (RRV) VP8* and cell surface glycoconjugates and possible inhibitors, in the present study, we have investigated the binding of a number of N-acetylneuraminic acid-based ligands (Figure 1) including Neu5Aca2Me, methyl S-(a-D-N-acetylneuraminosyl)-(2 ! 6)-6-thio-b-Dgalactopyranoside (Neu5Ac-a(2,6)-S-Galb1Me), methyl 9-O-acetyl-a-D-N-acetylneuraminide (Neu5,9Ac2a2Me), and the well-known sialidase inhibitor 2-deoxy-2,3-didehydro-DN-acetylneuraminic acid (Neu5Ac2en) in complex with RRV VP8* core by nuclear magnetic resonance (NMR) spectroscopy and computational chemistry
saturation transfer difference (STD) NMR experiments of Neu5Aca2Me in complex with RRV VP8* The 1H NMR and one-dimensional saturation transfer difference NMR (STD NMR) spectra of RRV VP8* in the presence of Neu5Aca2Me are shown in Figure 2A and B, respectively

Summary

Introduction

Rotaviruses are members of the Reoviridae family and are recognized as the single most important cause of severe gastroenteritis in the infants of a wide range of mammals including humans (Estes 2001). VP4 has been identified as the protein responsible for receptor binding and cell penetration (Ludert et al 1996; Zarate et al 2000; Arias et al 2002), the precise role of VP7 has not been completely defined, it has been proposed that it modulates the functions of VP4 (Beisner et al 1998) and interacts with cell surface molecules after the initial attachment of the virus through VP4 (Greenberg et al 1983; Coulson et al 1997; Mendez et al 1999). Both VP4 and VP7 independently elicit neutralizing antibodies and are determinants of virulence and induce protective immunity (Greenberg et al 1983)

Methods

Results

Conclusion