Figure 1.
Structure of the gD/Nectin-1 complex.
A. Schematic representations of human nectin-1 and HSV-1 gD. Signal peptides are shown as white boxes and transmembrane regions are shown as hatched boxes. Lollipops represent N-glycosylation sites. For nectin-1, numbering starts at methionine 1 of the open reading frame while for gD it starts at lysine 1 of the mature glycoprotein. Arrowheads indicate the location of truncations for production of nectin-1(346t), gD285t, gD306t and gD316t. Nectin-1 is colored in violet with a region previously implicated in gD binding colored in red. The gD Ig core is in shown in yellow, residues forming the HVEM binding hairpin are in green, residues 39 to 55 from the N-terminal extension are in dark grey, residues 185 to 250 from the C-terminal extension are in light gray and residues 251 to 316 are in red. B. Ribbon representation of the gD/Nectin-1 complex. The color code is the same as in Fig. 1A. The secondary structure elements are labeled as in Carfi et al. [21]. The β-strands are labeled according to the Ig V-fold. Unsolved loops in the distal portion of the nectin-1 C1 domain are drawn as dotted lines.
Figure 2.
The gD/Nectin-1 and Nectin-1/Nectin-1 interfaces.
A. Representation of the gD/Nectin-1 interface with key contact residues labeled and displayed in stick representation. Nectin-1 and gD are colored as in Fig. 1. B. Surface representation of gD showing the interface with nectin-1. gD is colored as in Fig. 1 with the nectin-1 contact area represented in blue and magenta. Mutations of residues colored blue have been shown to affect nectin-1 binding. Involvement of residues colored magenta has not previously been proposed. Most of the contacts involve residues from the gD C-terminal extension and, Y38 and Q27 from the N-terminal extension. C. Representation of a portion of the nectin-1 dimer interface (pdb-id 3ALP). Residues involved in key dimer interactions and that are involved in the gD/Nectin-1 interface are shown in stick representation. One monomer of nectin-1 is colored orange with black labels and the other is in pink with gray labels. D. Surface representation of the nectin-1 V-domain highlighting residues involved in either gD binding (magenta, left panel) or nectin-1 dimerization (orange, right panel).
Figure 3.
Effect of nectin-1 mutations on binding to gD.
A. Binding of purified gD(306t) to immobilized N1V-MBP by ELISA. Dilutions of gD were added to the same amount of immobilized N1V-MBP proteins. Bound gD was detected with polyclonal rabbit serum R8. B. Blocking of entry of HSV-1 KOS-tk12 into HeLa cells with soluble N1V-MBP proteins. Virus was preincubated with various concentrations of the indicated purified proteins for 1h and then added to HeLa cells. Entry was monitored 6h post infection by measuring β-galactosidase activity. The results are reported as percent of β-galactosidase signal compared to infection in the absence of soluble inhibitor (dotted line). A representative experiment is shown. C. Antigenic characterization of N1V-MBP mutants by ELISA. The purified wt fusion protein and each mutants were captured by the indicated immobilized anti-nectin-1 Ig and detected with anti-MBP antibody conjugated to HRP. Detection of each mutant is represented as percent of wt detection. An average of at least 5 experiments is shown with standard deviation.
Figure 4.
Effect of nectin-1 mutations on HSV entry. A
. Receptor negative B78H1 cells were transfected with full-length nectin-1 carrying the indicated mutations. HSV KOS-tk12 virus was added at various MOI. Entry was recorded 6h post infection by measuring β-galactosidase activity. A representative experiment is shown. Background signal from mock-transfected cells is also shown. B. Summary of all the entry data at an MOI of 5 pfu/cell. Levels of entry into cells expressing nectin-1 mutants are reported as the percentage of entry into B78H1 cells transfected with wt nectin-1. An average of 6 experiments is shown with standard deviation.
Figure 5.
Unliganded gD and gD/receptor complexes.
A, B. Surface representation of gD bound to nectin-1, with nectin-1 removed (A) or present (B). Residues 23–38 of gD are colored light green, the V-domain (residues 56–185) is colored yellow and the other residues from N-terminal (residues 39–55) and C-terminal (residues 186–250) extensions are in gray. Residues buried by nectin-1 in the gD/Nectin-1 complex are colored in dark blue. The V domain of nectin-1 is colored violet and the C1 domain is pink. C, D. View of gD bound to HVEM with HVEM removed (C) or present (D). Colored as in panel A with the N-terminal region involved in HVEM binding colored in dark green (residues 1 to 16) and in light-green (residues 17–38). Residues 1–16 of gD, which were not localized in the gD/Nectin-1 structure, fold back to form a hairpin structure when HVEM is bound. In the latter conformation, these residues mask the nectin-1 binding site. HVEM is shown in light blue. E. Conformation of unliganded gD colored as in panel A with residues 260–306 from the C-terminal extension colored in red.
Figure 6.
Similarities in the interactions between adhesion molecules with Ig-like fold and viral receptor-binding proteins.
The Ig-like domains of the receptors are shown in similar orientation and colored in red with the region that appears most structurally variable in cyan. A. Nectin-1 V-domain bound to HSV gD (gold). B. Coxsackie and Adenovirus receptor, CAR, domain D1 bound to Ad12 of adenovirus (blue) (pdb-id 1KAC). C. Junctional adhesin molecule-A, JAM-A, domain D1 bound to reovirus σ1 (green) (pdb-id 3EOY). D. Signaling lymphocyte activation molecule, SLAM, bound to measles virus hemagglutinin, MV-H, (purple) (pdb-id 3ALX).
Figure 7.
Functional pocket on the surface of gD.
Comparison between unliganded gD and gD/Nectin-1 complex. gD N-terminal residues 23–38 are green, the Ig-like V-domain is colored yellow (aa 56–185), the C-terminal region (aa 268–306) is shown in red and the remaining residues are white. A. In unliganded gD, the C-terminus of the ectodomain (red) is maintained in place by the insertion of Trp294 into a pocket made of the gD N-terminal residues, the α3-helix and residues from the Ig-core. This interaction is critical for function. B. The side chain of Phe129 (purple) located on the FG loop of the nectin-1 V-domain protrudes into the same pocket in the complex with gD. This binding configuration is not compatible with the native position of the gD C-terminus.