GII.13/21 Noroviruses Recognize Glycans with a Terminal β-Galactose via an Unconventional Glycan Binding Site

Figures

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  FIG 1. Saliva and glycan binding specificities of three GII.13 P proteins (Goulburn Valley/1983, 08N2045/2008, and SC1065/2016). (A) SDS-PAGE analysis of the P domain proteins of three GII.13 viruses, three GII.13 mutants, three GII.21 viruses, two GII.17 viruses, and a GII.14 virus. (B) Saliva-based binding assay showing binding signals (y axis) of the GII.13 proteins (0.5 μg/well) to type secretor samples (A, B, and O) and nonsecretor saliva samples (x axis). (C) Oligosaccharide-based binding assay showing that NoV GII.13 P proteins bind various synthetic polyacrylamide (PAA)-biotin-conjugated glycans in phosphate-buffered saline (PBS; pH 7.4). (D) Saliva-based binding assay of three GII.13 proteins to Lewis-positive nonsecretor samples and three kinds of Lewis-negative secretor samples, including A, B, and O. The experiment was repeated twice independently, and data are means and SDs (error bars) for absorbance values. OD450 nm, optical density at 450 nm.

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  FIG 2. Crystal structures of GII.13 SC1065/2016 P dimer in complex with Lec disaccharide (A to D) and GII.13 08N2045/2008 P dimer in complex with mucin core 2 trisaccharide (E to H). (A and E) Cartoon representation of SC1065/2016 P dimer with two protomers (light orange and green/cyan) in complex with Lec disaccharide (Galβ1-3GlcNAc) and 08N2045/2008 P dimer with one protomer (cyan) in complex with mucin core 2 trisaccharide [Galβ1-3(GlcNAcβ1-6)GalNAc]. Lec disaccharide and mucin core 2 trisaccharide are shown in stick representation. Galactose (Gal), yellow; N-acetylglucosamine (GlcNAc), gray; N-acetylgalactosamine (GalNAc), green. The oxygen and nitrogen atoms of the disaccharide and trisaccharide are shown in red and blue, respectively. (B and F) (2mFo-DFc) omit electron density maps of the Lec disaccharide (B) and mucin core 2 trisaccharide (F) in GII.13 SC1065/2016 and GII.13 08N2045/2008 P protein structures, respectively. The mesh map of Lec (B) was contoured at 1σ and that of mucin core 2 (F) was contoured at 1.5σ (blue) around the selection site, with a coverage at a 1.6-Å radius. (C) Network of hydrogen bond interactions between SC1065/2016 and Lec disaccharide, represented…

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  FIG 3. Glycan binding of the mutated GII.13 P proteins and GBS structural comparisons of GII.13/GII.21. (A) ELISA-based glycan binding assay of the 297Y and 361E and 297Y/361E mutations and native GII.13 SC1065/2016 P proteins. (B) Structure superposition of GII.13 SC1065 and GII.13 297 mutation based on GII.21 P dimer structure in complex with Lea (PDB code 4RM0). Lea is shown in stick representation. Gal, blue; GlcNAc, purpleblue; Fuc, yellow. The zoomed image shows structure superposition of two GII.13 proteins and GII.21 OIF as a cartoon representation. The P dimer interacts with Lea in the upper right image, and the bottom right is an enlargement at GII.13 297Y/N/GII.21 295Y and GII.13 G361/GII.21 E358 shown in stick representation, respectively. Hydrogen bond interaction between 295Y of GII.21 OIF and Gal or E358 and α-Fuc is represented by red dashed lines.

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  FIG 4. Glycan binding specificity of GII.21 and GII.17 P proteins for oligosaccharides. (A) ELISA-based assay of three GII.21 (OC06060/2006, YO284/2007, and ZS/2016) P proteins binding to oligosaccharides. (B) ELISA-based assay of two GII.17 (Kawasaki323/2014 and Kawasaki308/2015) P proteins to oligosaccharides. The experiment was repeated three times independently, and data are means and SDs (error bars).

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  FIG 5. Inhibition of the binding of GII.13/21/17 P proteins to glycans by nonfat milk. (A) Blocking by four concentrations (0%, 2%, 5%, and 10%) of nonfat milk of P proteins of GII.13 Goulburn Valley/1983, 08N2045/2008, and SC1065/2016 binding to Lec, Lac, or mucin core 2. The x axis shows synthetic PAA-conjugated Lec, Lac, or mucin core 2. (B) Blocking by four concentrations (0%, 2%, 5%, and 10%) of nonfat milk of P proteins of GII.21 OC06060/2006, YO284/2007, and ZS/2016 binding to Lec, Lac, or mucin core 2. (C) Binding of GII.17 Kawasaki323/2014 and Kawasaki308/2015 P proteins to blood type H disaccharide mixed with nonfat milk (0 to 10%).

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  FIG 6. Nonfat milk blocked the binding of GII.13/21/17 P proteins to saliva. (A) Two concentrations (0% and 2%) of nonfat milk blocked the binding of the P proteins of GII.13 Goulburn Valley/1983, 08N2045/2008, and SC1065/2016 to secretor (A, B, and O), and nonsecretor saliva. (B) Two concentrations (0% and 2%) of nonfat milk blocked the binding of the P proteins of GII.21 OC06060/2006, YO284/2007, and ZS/2016 to secretor (A, B, and O) and nonsecretor saliva. (C) Binding of GII.17 Kawasaki323/2014 and Kawasaki308/2015 P proteins mixed with nonfat milk (0 to 10%) to type secretor (A, B, and O) and nonsecretor saliva.

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  FIG 7. Inhibition of P protein-glycan interactions by free lactose. (A) Six concentrations of free lactose blocked binding of the GII.13 Goulburn Valley/1983, 08N2045/2008, and SC1065/2016 P proteins to synthetic PAA-conjugated Lec and mucin core 2 glycans. Synthetic PAA-conjugated Lac was included as a positive control. (B) Six concentrations of free lactose blocked binding of the GII.21 OC06060/2006, YO284/2007, and ZS/2016 P proteins to synthetic PAA-conjugated Lac, Lec, and mucin core 2 glycans. (C) Binding of the GII.17 Kawakasi323/2014 and Kawakasi308/2015 P proteins to blood type H disaccharide mixed with six concentrations of free lactose. The x axis shows the concentrations of free lactose. The experiment was repeated three times independently, and data are means and SDs (error bars).

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  FIG 8. Inhibition by free lactose of the binding of P proteins to saliva. (A) Four concentrations of free lactose blocked the binding of P proteins of GII.13 Goulburn Valley/1983, 08N2045/2008, and SC1065/2016 to secretor (A, B, and O) and nonsecretor saliva. (B) Four concentrations of free lactose blocked the binding of P proteins of GII.21 OC06060/2006, YO284/2007, and ZS/2016 to secretor (A, B, and O) and nonsecretor saliva. (C) Binding of P proteins of GII.17 Kawakasi323/2014 and Kawakasi308/2015 to type secretor (A, B, and O) and nonsecretor saliva. The x axis shows the free-lactose concentrations. The experiment was repeated three times independently, and data are means and SDs (error bars).

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  FIG 9. Structural comparison of GII.13 in complex with Lea, Lec, and mucin core 2 and models of NoV-glycan ligand interactions. (A) Structural superimposition of GII.13 VP1 (GenBank accession number BAQ94583) and SC1065/2016 08N2045/2008 in complex with Lea, Lec, and mucin core 2. Residues in the glycan binding site of GII.13 SC1065, GII.13 08N2045, and GII.13 VP1 are shown in stick representation in orange, yellow, and cyan. Amino acids residues of GII.13 VP1 in glycan binding site are in black. Lea, Lec, and mucin core 2 are shown in stick representation in pink, purple, silver, respectively. (B) Surface representations of GII.13 08N2045/2008, VP1, and SC1065/2016. GII.13 08N2045/2008, yellow; VP1, green; SC1065/2016, orange. The binding pocket in the GII.13 08N2045/2008, VP1, and SC1065/2016 glycan binding site is in blue, and the amino acid residues that interact with mucin core 2, Lec, and Lea are indicated. (C) Interaction networks between NoVs and glycan ligands: GI.1 (Norwalk virus)-H2 (PDB code 2ZL6), GII.4 (NSW0514)-B (PDB code 4OP7), and GII.13 (SC1065)-Lec. Residues in the glycan binding site of NoVs are shown in surface representation in cyan. The amino acid residues that…