Molecular basis of P[II] major human rotavirus VP8* domain recognition of histo-blood group antigens

Figures

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  Fig 1. Schematic representation of VP8* from P[II] genogroup with its corresponding binding glycan structures. (a) Schematic representation of P[8] VP8* and its binding glycan structures, Leb tetra-saccharide and LNDFH I. (b) Schematic representation of P[4] VP8* and its binding glycan structures, LNDFH I and LNFP I. (c) Schematic representation of P[6] VP8* and its binding glycan structures, LNT and LNFP I. (d) Schematic representation of P[19] VP8* and its binding glycan structures, LNT and LNFP I. The red boxes encircle Leb glycans and the blue boxes encircle non Leb glycans. (e) Inhibition of rotavirus Wa strain replication in HT29 cells. Left) Representative indirect immunofluorescence assay (IFA) microscopy image of P[8] RV Wa strain replication focuses in HT29 cells. Right) Quantitation of viral replication focus reductions observed in wells incubated with LNDFH1-BSA and H type 1-biotin-PAA in compared with cell culture wells with media only. Ley-PAA is a negative ligand control.

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  Fig 2. STD NMR analysis of Leb tetrasaccharide and LNDFH I glycan interactions with the P[8] VP8* domain. (a) NMR spectra of Leb tetra-saccharide in complex with P[8] VP8*. The bottom spectrum in (a) is 1H NMR reference spectrum of P[8] VP8* (33 μM) with Leb tetra-saccharide (1.86 mM). The middle spectrum in (a) is STD NMR spectrum of P[8] VP8* (33 μM) with Leb tetra-saccharide (1.86 mM). The protein was saturated with a cascade of 40 Gaussian-shaped pulses at 0.11 ppm, and the off-resonance was set to 50 ppm. The upper spectrum in (a) is the expansion of the STD NMR from 4.1 to 0.9 ppm. (b) The epitope mapping of Leb tetra-saccharide when bound to P[8] based on the STD effects: red, strong STD NMR effects (>30%); blue, medium STD NMR effects (20%-30%); green, weak STD NMR effects (<20%). (c) NMR spectra pf LNDFH I in complex with P[8] VP8*. The bottom spectrum in (c) is 1H NMR reference spectrum of P[8] VP8* (46 μM) with LNDFHI (2.3 mM). The middle spectrum in (c) is STD NMR spectrum of P[8] VP8* (46 μM) with LNDFHI (2.3 mM). The protein was saturated with a cascade of 40 Gaussian-shaped pulses at -0.25 ppm, and the off-resonance was set to 50 ppm. The upper spectrum in (c) is the…

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  Fig 3. STD NMR analysis of LNT and LNFPI glycan interactions with the P[6] VP8* domain. (a) NMR spectra pf LNT in complex with P[6] VP8*. The bottom spectrum in (a) is the 1H NMR reference spectrum of P[6] VP8* (46 μM) with LNT (4.6 mM).The middle spectrum in (a) is the STD NMR spectrum of P[6] VP8* (46 μM) with LNT (4.6 mM). The protein was saturated with a cascade of 40 Gaussian-shaped pulses at 0.63 ppm, and the off-resonance was set to 50 ppm. The upper spectrum in (a) is the expansion of the STD NMR from 4.3 to 2.3 ppm. (b) The epitope map of LNT when bound to P[6] based on the STD effects: red, strong STD NMR effects (>30%); blue, medium STD NMR effects (20%-30%); green, weak STD NMR effects (<20%). (c) NMR spectra pf LNFPI in complex with P[6] VP8*. The bottom spectrum in (c) is the 1H NMR reference spectrum of P[6] VP8* (46 μM) with LNFPI (2.3 mM). The middle spectrum in (c) is the STD NMR spectrum of P[6] VP8* (46 μM) with LNFPI (2.3 mM). The protein was saturated with a cascade of 40 Gaussian-shaped pulses at -0.21 ppm, and the off-resonance was set to 50 ppm. The upper spectrum is the expansion of the STD NMR from 4.4 to 1.0 ppm. (d) The epitope map of LNFPI when bound to…

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  Fig 4. NMR titration analysis of P[8] VP8* domain with Leb and LNDFH I glycans. (a) Chemical shift changes in P[8] VP8* [PDB ID: 2DWR] upon addition of Leb tetra-saccharide. The NMR data correspond to increasing ligand/protein ratios of 0:1 (red), 8:1 (orange), 18:1 (green), and 30:1 (blue). (b) Location of the large chemical shift changes on the P[8] VP8* surface [PDB ID: 2DWR] upon binding to Leb tetra-saccharide. Amino acids with chemical shift changes greater than 2.5σ or disappeared after titration are colored with red. (c) Ribbon Diagram shows the location of large chemical shift changes on the P[8] VP8* surface upon binding to Leb tetra-saccharide. The secondary structure is labeled. (d) Chemical shift changes in P[8] VP8* upon addition of LNDFH I. The NMR data correspond to increasing ligand/protein ratios of 0:1 (red), 6:1 (orange), 18:1 (green), and 25:1 (blue). The arrows show residues that have chemical shifts changes upon titration. Peaks that disappeared upon titration are circled. (e): Location of the large chemical shift changes on the P[8] VP8* surface [PDB ID: 2DWR] upon binding to LNDFH I. Amino acids with chemical shift changes greater than 1.5σ or disappeared after…

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  Fig 5. NMR titration analysis of P[6] VP8* domain with LNT and LNFP I glycans. (a) Chemical shift changes in P[6] VP8* [PDB ID: 6NIW] upon addition of LNT. The NMR data correspond to increasing ligand/protein ratios of 0:1 (red), 8:1 (orange), 18:1 (green), and 25:1 (blue). (b) Location of the large chemical shift changes on the P[6] VP8* surface [PDB ID: 6NIW] upon binding to LNT. Amino acids with chemical shift changes greater than 2σ or disappeared after titration are colored with red. (c) Ribbon Diagram shows the location of large chemical shift changes on the P[8] VP8* surface upon binding to Leb tetra-saccharide. The secondary structure is labeled. (d) Chemical shift changes in P[6] VP8* upon addition of LNFP I. The NMR data correspond to increasing ligand/protein ratios of 0:1 (red), 8:1 (orange), 18:1 (green), and 35:1 (blue). The arrows show residues that have chemical shifts changes upon titration. Peaks that disappeared upon titration are circled. (e) Location of the large chemical shift changes on the P[6] VP8* surface [PDB ID: 6NIW] upon binding to LNFPI. Amino acids with chemical shift changes greater than 1.5σ or disappeared are colored with red. (f) Ribbon Diagram shows…

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  Fig 6. NMR titration analysis of P[8] VP8* mutants with LNDFH I glycan. (a) Chemical shift changes in T78A P[8] VP8* upon addition of LNDFH I. The NMR data correspond to increasing ligand/protein ratios of 0:1 (red), 8:1 (orange), 12:1 (green), and 16:1 (blue). (b) Location of the large chemical shift changes on the T78A P[8] VP8* surface upon binding to LNDFH I. (c) Ribbon Diagram shows the location of large chemical shift changes on the T78A P[8] VP8* surface upon binding to LNDFH I. (d) Chemical shift changes in D79A P[8] VP8* upon addition of LNDFH I. The NMR data correspond to increasing ligand/protein ratios of 0:1 (red), 8:1 (orange), 12:1 (green), and 16:1 (blue). (e) Location of the large chemical shift changes on the D79A P[8] VP8* surface upon binding to LNDFH I. (f) Ribbon Diagram shows the location of large chemical shift changes on the D79A P[8] VP8* surface upon binding to LNDFH I. (g) Chemical shift changes in K168A P[8] VP8* [PDB ID: 2DWR] mutant upon addition of LNDFHI. The NMR data correspond to increasing ligand/protein ratios of 0:1 (red), 8:1 (orange), 12:1 (green), and 16:1 (blue). (h) Location of the large chemical shift changes on the K168A P[8] VP8* surface…

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  Fig 7. HADDOCK docking results of P[8] with its glycans. (a) Cartoon figures in the left panel show the superposition of the top five best-scoring Leb tetra-saccharide bound P[8] VP8* structures. The right panel in (a) highlights the binding pocket of P[8] in recognizing Leb tetra-saccharide. (b) The superposition of the top five best-scoring LNDFH I bound P[8] VP8* structures is shown in the left panel. The right panel in (b) highlights the binding pocket of P[8] in recognizing LNDFH I. Red colors in the proteins represent the binding interface. Different colors were used to represent different carbohydrate moiety of the ligand: Lewis fucose (magenta), secretor fucose (green), galactose (yellow), N-acetylglucosamine (blue), glucose (cornflower blue).

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  Fig 8. Crystal structure of LNFP I-P[6] complex. (a) The surface model of LNFP I-P[6] complex [PDB: 6OAI]. (b): Ribbon diagram shows the LNFP I-P[6] VP8* complex. The right panel in (b) highlights the binding pocket of P[6] VP8* in recognizing LNFP I. Red represents the binding interface. Different colors were used to represent different carbohydrate moiety of the ligand: Green colors the secretor fucose residue, yellow colors the Galactose residue, blue colors the N-Acetylglucosamine residue, cornflower blue colors the Glucose residue.

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  Fig 9. Structure-based sequence alignment of VP8* domains. The VP8* sequences of RVs with known binding sites are included. The VP8* sequences of P[19], P[6], P[4] and P[8] in P[II] genogroup, P[3], P[7] in P[I] genogroup, P[14] in P[III] genogroup and P[11] in P[IV] genogroup are included and the amino acids corresponding to the binding site are highlighted in red. Yellow highlights the potential binding site of Wa and other strains of P[8] VP8* in recognizing HBGAs.

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  Fig 10. The binding interface of different genotypes of VP8* in recognizing their corresponding glycans. (a) Surface-rendered structure of the apo P[6] VP8* domain [PDB ID: 6NIW] as a representative of the VP8* structures depicting a summary of the distinct glycan binding sites identified to date. The red-colored region indicates the ββ binding motif that P[3]/P[7] use in binding sialic acid, P[14] in binding type A HBGAs, P[11] in binding type 1 HBGAs, and P[8] in recognizing Lewis type HBGAs. The blue-colored region indicates the βα binding pocket that the P[19], P[6] and P[4] RVs use in recognizing H-type 1 HBGAs. (b) Detail of the sialic acid binding interface (red) of P[3] [PDB ID:1KQR]. (c) Detail of the LNT binding site of P[11] [PDB ID: 4YFZ]. (d) Detail of the LNDFH I binding interface of P[8]. (e) Detail of the LNFP I binding site of P[4] [PDB ID: 5VX5].