Despite the discovery of human norovirus nearly 40 years ago (27
), little is known about the capsid interaction with ligands (18
) other than HBGAs (8
). Our finding that citrate binds at the terminal fucose binding site was somewhat unexpected, given that the structure of citrate is unlike the structure of fucose and considering that the GII.10 P domain could not bind HBGAs having an α-fucose1-3/4 saccharide (21
). In an earlier enzyme immune assay study, Feng et al. screened ~5,000 compounds (the Diversity screening set; Timtec, Inc.) for their ability to block GI and GII norovirus virus-like particles (VLPs) from binding to saliva samples of known HBGA type (18
). They found 14 compounds that had strong inhibition; however, the mode of action was not determined. In a more recent NMR study, Rademacher et al. screened ~500 compounds (the Maybridge Ro5 fragment library; Thermo Fisher Scientific, Inc.) for their ability to bind to a GII.4 VLP HBGA binding site (44
). They showed that both univalent and multivalent compounds were capable of binding to the HBGA binding site. Interestingly, for both studies, the compounds that showed the highest affinities included compounds with at least one ring component. Taken together, these studies indicated that the HBGA binding site was capable of binding numerous compounds other than HBGAs, ranging from the small (smallest) citrate molecule to larger multivalent compounds.
For over a decade, the GII.4 noroviruses have remained as the dominant genotype of outbreaks of gastroenteritis around the world and as such the most well studied. Most studies agreed that a dominant GII.4 norovirus was replaced the following year or next by a new GII.4 “variant” norovirus that had ~5% amino acid change in the capsid gene (6
). The reason that the GII.4 variants dominated and not some other genotype was unknown, but studies have shown specific mutations at or surrounding the HBGA binding site were capable of altering the HBGA binding patterns (15
). These small changes were thought to lead to new GII.4 variants capable of causing pandemics, analogous to influenza A virus evolution (14
). Despite these amino acid changes, few if any occurred at the fucose-binding site, thus highlighting the common site of vulnerability for GII noroviruses, especially for the pandemic GII.4 variant noroviruses. It is not known if the GI noroviruses will bind citrate given that the GI and GII P domain interactions with HBGAs were different, but since GI.1 P domain interacted with α-fucose1-2 and it was reported that the HBGA binding site was conserved among GI noroviruses (12
), we suspect that GI noroviruses may also bind citrate, although further structural studies are needed.
Our unexpected finding that citrate and fucose have similar binding modes to the norovirus GII.10 P domain raises the question of whether such citrate mimicry of monosaccharide binding could be a general phenomenon or whether it is specific to norovirus and other caliciviruses. To investigate this, we performed in silico
docking studies of citrate against four different fucose-binding proteins (Anguilla anguilla
agglutinin, Aleuria aurantia
lectin, Streptococcus pneumoniae
virulence factor SpGH98, and Pseudomonas aeruginosa
PA-IIL lectin) and two other saccharide-binding proteins (parainfluenza virus 5 hemagglutinin-neuraminidase and porcine adenovirus type 4 galectin domain), for which fucose or other saccharide-bound crystal structures were available (see Table S1 in the supplemental material). Computational docking analyses reveal different levels of citrate mimicry of monosaccharide binding for other saccharide-binding proteins. For Anguilla anguilla
agglutinin, citrate, in its predicted binding pose, overlapped with the C-5, C-4, C-3, O-5, O-4, and O-3 atoms of fucose in a similar way to what was observed in the GII.10 P domain (Table S1), while forming hydrogen bonds with the same sets of protein residues as fucose (see Fig. S4 in the supplemental material). Citrate was thus predicted to show a high degree of mimicry to fucose, similarly to our experimental findings for the GII.10 P domain. For the other three fucose-binding proteins, citrate, in its predicted binding poses, did not overlap with the cocrystallized fucose, although it still formed the same sets of polar interactions as the cocrystallized fucose (see Fig. S5 to S7 in the supplemental material). Hence, our docking studies suggest that the mimicry between citrate and fucose binding observed for the GII.10 P domain could be a common, although not universal, phenomenon across other fucose-binding proteins. For all six fucose- and other saccharide-bound proteins for which docking was performed, the predicted citrate binding poses were able to form polar interactions with the same sets of protein residues as the cocrystallized ligand see (Fig. S4 to S9 in the supplemental material), indicating that citrate might be generally useful as a scaffold for designing glycomimetic inhibitors against these and other saccharide-interacting pathogens. Furthermore, a search of the ZINC database (4
) revealed that there are more than three thousand compounds with at least 50% similarity to citrate. Thus, in silico
screening of this database may present a promising approach for identifying small molecules that bind to saccharide-binding proteins. We note, however, that the predicted binding pose of citrate docked to fucose-bound GII.10 P domain had a root mean square deviation (RMSD) of 3.60 Å, while the predicted binding pose of citrate docked to citrate-bound GII.10 P domain with the cocrystallized water molecule had an RMSD of 1.87 Å. This indicates that the resulting docking modes could be error prone. Given that calculating small molecule-receptor binding energies is a difficult and error-prone task (24
), ultimately experimental validation would be necessary to confirm the generality of the citrate-saccharide mimicry predicted here.
The STD NMR data provided strong evidence that the integrity of the GII.10 P domain remained unchanged in the presence of different concentrations of citrate buffer and since the pH of the citrate buffer remained more or less the same during the titration, a specific effect of citrate was responsible for the reduction in HBGA attachment. Although the KD
values of citrate and H type 2 trisaccharide for the GII.10 P domain are in the range of 360 to 490 μM, these relatively weak affinities are typical for univalent protein-carbohydrate interactions (17
). Given that 90 copies of dimeric P domains are present on norovirus capsid, it is plausible that a multivalent version of citrate- or fucose-like ligands would greatly enhance affinities and provide a starting point for norovirus inhibitors. Indeed, Rademacher et al. show that multivalent fucose-like compounds have increased avidity over their univalent counterparts (44
In conclusion, we have described the structural basis by which citrate binds to the HBGA binding site of the norovirus GII.10 P domain and can in turn inhibit HBGA binding. Natural compounds, such as juice from lemons and limes, which contain ~300 mM citric acid (42
), may already reduce or inhibit norovirus infections, as suggested by a number of recent studies (23
). In regard to this, it is tempting to speculate that a few drops of lemon juice with one's oysters might reduce norovirus infection. Epidemiological studies on the ingestion of foods high in citrate and norovirus infection may be illuminating, as may be correlations with related glycomimetics—e.g., with ascorbic acid (vitamin C). Controlled possibly volunteer studies should also provide an accurate assessment of norovirus inhibition. Additional compound screening will likely be required to identify a universal norovirus inhibitor with high potency and broad reactivity, and the structural basis for norovirus interaction with citrate as revealed here may be helpful in such efforts.