A ligand-binding pocket in the dengue virus envelope glycoprotein

Abstract

Dengue virus is an emerging global health threat. Its major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. A crystal structure of the soluble ectodomain of E from dengue virus type 2 reveals a hydrophobic pocket lined by residues that influence the pH threshold for fusion. The pocket, which accepts a hydrophobic ligand, opens and closes through a conformational shift in a beta-hairpin at the interface between two domains. These features point to a structural pathway for the fusion-activating transition and suggest a strategy for finding small-molecule inhibitors of dengue and other flaviviruses.

Fig. 1.

Dengue E protein and its ligand-binding pocket. (A) Domain definition of dengue E. Domain I is red, domain II is yellow, and domain III is blue. (B) The dengue E protein dimer, colored as in A, in complex with β-OG. The β-OG, shown in green, is bound in a hydrophobic pocket under the kl hairpin. A putative receptor-binding loop in domain III (residues 382–385) is marked with a triangle. The glycans in domains I and II are shown in a ball-and-stick representation in red and yellow, respectively. Disulfide bridges are shown in orange. (C) Enlargement of the kl hairpin region, with the structure of dengue E in the absence of β-OG (in gray) superimposed. The strands of the kl hairpin are labeled with “o” or “c” subscripts for the open (β-OG-bound) and closed forms, respectively. The β-OG molecule, shown in a space-filling representation, occupies the ligand-binding pocket. (D) Superposition of the structures of dengue E and TBE E, both in the absence of β-OG. Dengue E is colored as in B, and TBE E is in gray. The view is the same as in C. Figs. 1, 2, and 3 were generated with bobscript (33, 34) and raster 3d (35).

Fig. 2.

The glycan at residue 153 in dengue 2 virus E protein. (A) The E protein dimer, viewed perpendicular to the dyad axis (and the view in Fig. 1 A). Both glycans are approximately perpendicular to the viral surface. Domain I and the attached glycan are shown in red, domain II and the attached glycan are shown in yellow, and domain III is in blue. Disulfide bridges are shown in orange. The molecule of β-OG bound in the hydrophobic pocket underneath the kl hairpin is in green. A putative receptor-binding loop in domain III (residues 382–385) is marked with a triangle. (B) Enlargement of the area surrounding the glycan at residue 153 in domain I, with the structure of TBE envelope protein superimposed (gray) onto domain I of dengue virus E protein. The fusion peptide is highlighted in orange. The disulfide bridge between residues 92 and 105 is shown in green.

Fig. 3.

Mutations affecting the pH threshold of fusion (or virulence) in flaviviruses (–41). The mutated residues line the interior of the ligand-binding pocket. For unconserved residues, the residue type in the virus in which the mutation was identified is listed first, followed by the residue type in dengue 2. The coloring is the same as in Fig. 1.

Fig. 4.

Proposed subunit packing interactions in various flaviviral icosahedral assemblies. (A) Suggested transition from the previously studied T = 1 subviral particles (12) to the fusion-competent T = 1 particle at low pH. On acidification, domain II is proposed to swing out about a hinge at the domain I/II interface, creating homotrimeric contacts at the threefold axis. Clusters of three fusion peptides are displayed at the tip of each trimer. (B) The packing in T = 3 virus-like particles deduced from image reconstructions of dengue virions (13). The 180 subunits are not related by local threefold symmetry. (C) Suggested T = 3 packing intermediate for the virion at low pH (13). E is shown in its native (high pH) conformation. Because all monomers are related by local threefold symmetry, the low-pH conformational change will result in the formation of trimers, as in A.