Protein Preparation and Nectin-4 binding to CDV-H.

A. Schematic images for expression region of CDV-H globular domain (top), dog SLAM ectodomain (middle) and dog Nectin-4 (bottom). CT, cytoplasmic tail; TM, transmembrane; CO, connector region; SP, signal peptide. B. SDS-PAGE for CDV-H protein in reducing (left) and non-reducing (right) conditions. The arrows indicate monomer (left) and disulfide-linked dimer (right) of CDV-H. C. Gel filtration chromatogram of CDV-H using Superdex 200 column. The arrow indicates the peak of CDV-H. D. Surface plasmon resonance analysis for the interactions between CDV-H and Nectin-4 receptors: dog (left) and human (right) Nectin-4. The Green, Red and Blue lines indicate CDV-Hwt, CDV-Hvac and β2 microglobulin as a negative control, respectively.

Crystal structure of CDV-H.

A. Crystals of CDV-H head domain. B. Overall structure of CDV-H with secondary structures in cartoon model, colored gradually from blue (N-terminus) to red (C-terminus). C. Structural comparison of CDV-H (rainbow color) and MeV-H (grey). D. Electron density map (2.5 σ omit map shown in black mesh) around Asn422 of CDV-H attached with N-glycan.

Homodimer structure and its interface of CDV-H.

A. Homodimer structure of CDV-H with secondary structures in cartoon model as the same as Fig. 2B (rainbow: chain A and grey: chian B). The black arrows indicate the receptor binding sites. B. Structural comparison of CDV-H with other paramyxovirus hemagglutinin dimers. MeV-H (top, PDB code: 2ZB6), PIV5-HN (middle, PDB code: 4JF7) and NDV-HN (bottom, PDB code: 3T1E) are shown in similar direction with CDV-H of Fig. 3A. C. The closed-up images for three sites at the homodimer interface. One of the CDV-H dimer is shown in slate, asymmetric unit in grey. One of the MeV-H dimer is colored hot pink.

Putative SLAM binding site on CDV-H.

A. Structures of CDV-H with putative SLAM binding sites (left, slate) and SLAM complex of MeV-H (right, hot pink) are shown. B. The residues that are involved in SLAM receptor binding revealed by mutagenesis studies are shown in stick model on CDV-H (top) and MeV-H (bottom). C. Amino acid sequence alignment of v-type domains of SLAMs. Asterisks (*) indicate fully conserved residue. A colon (:) indicates conservation between groups of strongly similar properties. A period (.) indicates conservation between groups of weakly similar properties. Interacting with hemagglutinin residues (<4.0 Å) are indicated in yellow-colored boxes.

Putative Nectin-4 binding site on CDV-H.

A. Structures of CDV-H with putative Nectin-4 binding sites (left, slate) and Nectin-4 complex of MeV-H (right, hot pink) are shown. B. The residues that are involved in Nectin-4 receptor binding revealed by mutagenesis studies are shown in stick model on CDV-H (top) and MeV-H (bottom). C. Amino acid sequence alignment of v-type domains of Nectin-4s. Abbreviations and green-colored boxes are shown in the same manner with Figure 4C.

Glycan shield of CDV-H.

A. Schematic images of full-length and expression constructs of ectodomain of CDV-Hs (wild-type and vaccine strains), together with the of MeV-H. B. Mutagenesis studies for all potential glycan modification revealed to show that N390D and N630D showed clear western blot data. C. Differences in glycan shields. Glycosylation sites on the surface of CDV-H (left) and MeV-H (right) dimers shown in white, with common N-linked glycans modeled in black and deep blue. Receptor binding sites were shown in yellow circle on the CDV-H dimer. Tamarin SLAMs bound to the MeV-H dimer were shown in yellow. Two-fold symmetry axis was shown as black ellipse.

Structural Dynamics of CDV-H analyzed by HS-AFM.

A. Homodimeric structure of CDV-H with a part of stalk region (residues 149 to 604). The globular head domain (grey) and the stalk region (magenta) are shown in cartoon model. B. Serial HS-AFM images of CDV-H homodimer at 2 fps are shown. Scale bar indicates 20 nm length. Graphs under each HS-AFM images indicate the z-axis parameter between red and white triangle. C. The distances between signal centers (Dcc) of representative homodimer were monitored at time scales. D. The numbers of CDV-H homodimers are classified as several distances.

Proposed model of membrane fusion at CDV entry.

Step1. The homodimer dimers have stalk regions (colored orange) attached to fusion proteins. Each H protein dimers is bridged by disulfide bond (shown in red bar) at “neck” region. Step2. Binding of receptor (SLAM or Nectin-4) to the head domains. Steps 3 and 4. Receptor pulling causes the head domains to dissociate a dimer of homodimers and completely collapse to individual domains. Step5 and 6. Fusion proteins are allowed to access the membrane of target cells and complete fusion.

Purification of dog and human soluble Nectin-4.

A. Gel filtration chromatogram of dog (left) and human (right) Nectin-4 using Superdex 200 column. The arrow indicates the peak of each Nectin-4. B. SDS-PAGE for dog (left) and human (right) Nectin-4 in reducing conditions. The arrow indicates the band of each Nectin-4.

Structure-based alignment of receptor binding glycoproteins from various paramyxoviruses.

Attachment glycoproteins were aligned with the CDV-H structure. G proteins of Hendra virus; HeV (PDB code: 2VSK) and Nipah virus; NiV (PDB code: 3D12), HN proteins from Mumps virus; MuV (PDB code: 5B2C), Parainfluenza virus 5; PIV5 (PDB code: 4JF7), Newcastle disease virus; NDV (PDB code: 3T1E), Sosuga virus; SosV (PDB code: 6SG8), and human parainfluenza virus 3; hPIV3 (PDB code: 1V2I);, H protein of MeV (PDB code: 2ZB6). Green, yellow, and gray boxes indicate α-helices, β-sheets, and disordered regions in the structure, respectively. Red letters indicate the residues involved in homodimer formation. The positions of the secondary structures of the six-bladed β-propeller CDV-H are shown underneath the alignment.

The superposition of CDV-H onto the MeV-H complex structure.

The superposition of CDV-H onto the MeV-H-tamarin SLAM complex (left), and onto MeV-H-hNectin-4 complex (right). Coloring is the same as that in Figure 4 and 5.

Glycan modifications onto CDV-H and MeV-H-SLAM complex.

A. Side view of glycan models (five residues) mapped on the surface presentation of CDV-H (left) and MeV–H-SLAM complex structure (right) was represented in the same manner with Fig. 6C. B. Schematic presentation of CDV-H dimer showing the locations of glycosylated (green) and unglycosylated (red) asparagine. RBSs was shown in yellow.