Structural basis of glycan specificity of P[19] VP8*: Implications for rotavirus zoonosis and evolution

Figures

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  Fig 1. Binding of P[19] VP8* to LNFP I and mucin core 2. (a) A dose dependent binding of P[19] VP8* to the type I HBGA pentasaccharide Lacto-N-fucopentaose I (LNFP I) and mucin core 2. As a control, P[14] VP8* that specifically binds to the A-HBGA was included. Each condition was tested with three replicates. The error bars represent the standard deviation. (b) Schematic representation of the glycan structures. Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Fuc, fucose.

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  Fig 2. Comparison of ligand binding sites among P[3], P[14], P[11] and P[19] RVs with their specific ligands. (a) Schematics of known ligand binding sites based on the P[19] VP8* structures with different colors. The detailed ligand binding sites for each genotype are shown in (b)–(e). P[3] and P[14] shared a common binding site locating in one corner of the cleft (red color) to interact with the sialic acid or A antigen of the two genotypes, respectively (a)-(c). The ligand binding site of P[11] is also located in the cleft region (green color) while the cleft is wider and its ligand spread the entire cleft (d). P[19] characterized in our current study also has a wider cleft but the ligand binding site (blue color) is in a totally new location different from the other genotypes (e). Arrows indicate relative widths of cleft for individual genotypes.

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  Fig 3. Structure of P[19] VP8* in complex with LNFP I. (a) Surface representation of P[19] VP8* (grey) with a bound LNFP I. The penta-saccharide LNFP I is shown in a ball-and-stick representation (yellow) with the nitrogen and the oxygen atoms being colored in blue and red, respectively. The amino acid residues in the P[19] VP8* which participate in hydrogen bonds and hydrophobic interactions with LNFP I are in blue color. The GlcNAc and an inner Gal moiety of the LNFP I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc) insert into two adjacent well-defined pocket in the P[19] VP8* structure. The Fuc projects out from the VP8* binding surface and does not have any direct contact with VP8*. (b) Network of hydrogen bond interactions (dashed lines) between the VP8* residues and LNFP I. The interaction residues are shown in stick model and the glycan is shown in stick representation with different colors, Fuc colored in green, Gal in yellow, GlcNAc in purple and Glc in pink. Participating water molecules are shown as small spheres (orange). Molecular interactions of LNFP I with P[19] VP8* were analyzed using LIGPLOT (see S4 Fig).

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  Fig 4. Structure of P[19] VP8* in complex with mucin core 2. (a) Surface representation of P[19] VP8* structure with bound mucin core 2 following the same coloring scheme as in Fig 2a. (b) Network of hydrogen bond interactions between the VP8* residues and mucin core 2 with the same coloring scheme as in Fig 2b. All the three sugar moieties of mucin core 2 are involved in interaction with the VP8*, with the GlcNAc and GalNAc of mucin core 2 (GlcNAcβ1-6GalNAcβ1-3Gal) binding to the two adjacent well-defined pocket on the surface of P[19] VP8*.

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  Fig 5. Structural comparison of P[19] VP8* in complex with LNFP I and mucin core 2. (a) Superimposition of P[19] VP8* complexes with LNFP I (dark blue) and mucin core 2 (red). Amino acid residues of the P[19] VP8* which participate in interactions with the two glycans are indicated in light blue. (b) Schematic interaction diagram of P[19] VP8* with the two glycans. Ten amino acids interacted with both glycans with two more (T184 and T21) interacted with LNFP I.

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  Fig 6. Inhibition of P[19] RV replication in cell culture by LNFP I and mucin core 2. LNFP I shows significant dose-dependent inhibition. Higher doses are required for mucin core 2 and adding of both LNFP I and mucin core 2 resulted in the highest inhibition. Error bars represent standard errors from triplicate repeats and the experiment was repeated once. The statistical significance was calculated by ANOVA, the asterisk refers to statistical difference <0.05.

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  Fig 7. Structure-based sequence alignment of VP8*s. The VP8* sequences of P[19] and other P[II] (P[4], P[6] and P[8]) and two P[I] RVs (P[10] and P[12]) are included and the amino acids corresponding to the binding site of P[19] VP8* are highlighted in purple. The VP8* sequences of other RVs with known binding sites are also included. The amino acids of their binding sites are highlighted in red for P[3] and P[7] that recognize the sialic acids and P[14] that recognizes the A-HBGAs. The amino acids of the binding site of P[11] RVs are highlighted in green that recognizes the poly-linker type 2 HBGA precursors.

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  Fig 8. Superimposition of LNFP I-bound P[19] VP8* with apo VP8*s of P[4] and P[8] RVs. (a) The VP8* structure of P[4] (PDB ID: 2AEN) (cyan) and P[19] VP8* (grey) with LNFP I as colored sticks. The zoomed-in inset panel at the right shows a detailed view of the LNFP I binding interface defined by the P[19]-LNFP I complex structure. The structural overlay highlights the differences in the side chain orientations between the P[19] VP8-LNFP I-bound structure and apo P[4] VP8 structure. Residues Y169 and R209 in the apo P[4] VP8* tilt away from the binding pocket, which may destabilize the interaction with LNFP I. (b) Superimposition of the VP8* of P[8] (PDB ID: 2DWR) (pink) and the LNFP I-bound P[19] VP8*(grey) with the zoomed-in inset at right showing the detailed view of the ligand binding site in the P[19]-LNFP I complex structure. The side chain of R172 in P[8] VP8* sterically clashes with the oxygen atom of the Gal-IV moiety of LNFP I, providing a possible explanation for lack of P[8] VP8* binding to LNFP I (26).