Research ArticleVIROLOGY

Crystal structure of bovine herpesvirus 1 glycoprotein D bound to nectin-1 reveals the basis for its low-affinity binding to the receptor

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Science Advances  13 May 2020:
Vol. 6, no. 20, eaba5147
DOI: 10.1126/sciadv.aba5147
  • Fig. 1 BHV-1 gD engages nectin-1 with low binding affinities.

    (A) A schematic view of BHV-1 gD. The signal peptide (SP), ectodomain, transmembrane domain (TM), and cytoplasmic domain (CP) were marked with the boundary residue numbers. For recombinant expression of BHV-1 gD in insect cells, the protein was truncated (amino acids 1 to 342 for gD342, amino acids 1 to 301 for gD301, and amino acids 1 to 274 for gD274), engineered with a GP67 signal peptide for secretion, and added with a C-terminal 6×His tag for purification. (B) Representative size-exclusion chromatographs of gD342, gD301, and gD274. The separation chromatograph of the protein and the SDS–polyacrylamide gel electrophoresis analyses of the pooled samples were shown. Lane 1: The pooled sample collected from the first half of the protein elution peak. Lane 2: Sample collected from the second half of the peak. (C to E) An SPR assay characterizing the binding kinetics between BHV-1 gD and nectin-1 IgV. Gradient concentrations of BO- or HU-nectin-1 IgV were flowed through gD342, gD301, and gD274 immobilized on the chip surface. The real-time binding profiles are recorded and shown.

  • Fig. 2 Structure of free BHV-1 gD.

    (A) Cartoon representation of the overall structure. The extracellular domain of BHV-1 gD includes an IgV-like core (magenta) and N- and C-terminal extensions (yellow and cyan, respectively). The secondary structural elements and three disulfide bonds are labeled. (B) Comparison of HSV-1 (yellow), HSV-2 (salmon), PRV (cyan), and BHV-1 (blue) gD structures. The variant secondary structure elements are marked. (C to E) Topology diagrams of BHV-1 (C), PRV (D), and HSV-1 and HSV-2 (E) gDs. The figures are made on the basis of our BHV-1 gD structure, and the structures of PRV, HSV-1, and HSV-2 gDs with Protein Data Bank (PDB) codes of 5X5V, 1L2G, and 4MYW, respectively. The α helices and β strands are individually shown as rectangles and arrows, and those exhibiting differences are highlighted in orange. (F) Structure-based sequence alignment for BHV-1 gD and its homologs. The spirals and arrows indicate α helices and β strands, respectively. The cysteine residues forming disulfide bonds are marked numerically. The C″ strand, G/α2 interloop, and PxxW motif are highlighted with black, red, and magenta dotted rectangles, respectively. Residues of BHV-1, PRV, HSV-1, and HSV-2 gDs (19, 21) interfacing with nectin-1 are marked with black circles, boxes, stars, and triangles, respectively.

  • Fig. 3 Structure of BHV-1 gD bound to BO-nectin-1 IgV.

    (A) An overview of the complex structure. The IgV-like core and the N- and C-terminal extensions of BHV-1 gD and the nectin-1 receptor are colored magenta, yellow, cyan, and green, respectively. The left panel is rotated around a vertical axis for about 160° to generate the right one. BO-nectin-1 IgV in the left panel and gD in the right panel are shown in surface representation. (B) Structural comparison of the BHV-1 gD structures in the free (gray) and the nectin-1 bound form [colored as in (A)]. The N-terminal loop and the G/α2 interloop that experience conformational change upon receptor binding are shaded. (C to F) Homologous nectin-1 binding mode for alphaherpesviral gDs. (C) Structure of BHV-1 gD bound to BO-nectin-1. (D) Structure of PRV gD bound to swine nectin-1 (PDB code: 5X5W). (E) Structure of HSV-1 gD bound to HU-nectin-1 (PDB code: 3U82). (F) Structure of HSV-2 gD bound to HU-nectin-1 (PDB code: 4MYW). The complex structures are oriented similarly and aligned in parallel. The coloring scheme follows that in (A).

  • Fig. 4 The atomic interaction details at the BHV-1-gD/BO-nectin-1 interface.

    (A) An overview of the binding interface. The nectin-1 receptor and the gD ligand are shown in surface and cartoon representations, respectively. The interface components in BHV-1 gD, including the N-loop and the multiple elements in the C-terminal extension, are highlighted and marked with patch numbers 1 and 2. The amino acid interaction details for each patch are further delineated in (B) and (C), respectively. (B) The interaction of gD N-loop with nectin-1. (C) The interaction of the gD C-extension elements with nectin-1. For clarity, only those residues providing ≥2 vdw contacts are shown and labeled. A full list of the interface residues are summarized in tables S3 and S4. Dashed lines indicate H-bonds. (D) Sequence alignment for the IgV domain of BO-nectin-1 and HU-nectin-1. The residues interfacing with BHV-1 gD are marked with black stars.

  • Fig. 5 R188 in the G/α2 interloop interferes with nectin-1 engagement.

    (A) A magnified view on the G/α2 interloop in the free gD structure (gray) and in the nectin-1–bound complex structure (magenta). The nectin-1 receptor is colored green. The residues referred to in the text are shown and labeled. The approximate 6.8-Å shift observed with R188 and about 90° rotation with Y190 are highlighted. (B and C) SPR experiments characterizing the binding of gD R188G (B) and Y190G (C) mutants to BO-nectin-1 IgV. The representative kinetic profiles are shown. (D) Structural comparison of HSV-1 gD in the free (gray) and nectin-1–bound forms (magenta) and of HSV-2 gD in the free (yellow) and nectin-1–bound forms (green). (E) Structural comparison of PRV gD in the free (gray) and nectin-1–bound forms (cyan).

  • Table 1 Statistics of the kinetic binding affinities between BHV-1 gD and nectin-1 IgV determined by SPR.

    The fast-on/fast-off kinetic data are analyzed by the steady-state affinity model. The results from three independent experiments are listed.

    Binding pairKD (M)Average KD (M)SD for KDModel
    gD274 to HU-nectin-1-IgV7.575 × 10−77.008 × 10−70.676 × 10−7Steady-state affinity
    6.260 × 10−7
    7.188 × 10−7
    gD274 to BO-nectin-1-IgV6.752 × 10−74.987 × 10−71.565 × 10−7Steady-state affinity
    4.437 × 10−7
    3.771 × 10−7
    gD301 to HU-nectin-1-IgV7.648 × 10−78.792 × 10−71.008 × 10−7Steady-state affinity
    9.183 × 10−7
    9.546 × 10−7
    gD301 to BO-nectin-1-IgV2.591 × 10−73.406 × 10−71.061 × 10−7Steady-state affinity
    3.022 × 10−7
    4.606 × 10−7
    gD301-R188G to BO-nectin-1-IgV7.908 × 10−87.054 × 10−81.352 × 10−8Steady-state affinity
    5.495 × 10−8
    7.758 × 10−8
    gD301-Y190G to BO-nectin-1-IgV8.067 × 10−77.502 × 10−71.648 × 10−7Steady-state affinity
    8.974 × 10−7
    5.646 × 10−7

Supplementary Materials

  • Supplementary Materials

    Crystal structure of bovine herpesvirus 1 glycoprotein D bound to nectin-1 reveals the basis for its low-affinity binding to the receptor

    Dan Yue, Zhujun Chen, Fanli Yang, Fei Ye, Sheng Lin, Bin He, Yanwei Cheng, Jichao Wang, Zimin Chen, Xi Lin, Jing Yang, Hua Chen, Zhonglin Zhang, Yu You, Honglu Sun, Ao Wen, Lingling Wang, Yue Zheng, Yu Cao, Yuhua Li, Guangwen Lu

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    • Figs. S1 to S4
    • Tables S1 to S4

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