Figure 1.
(A) Ribbon diagram of the subunit of the E2s dimer, side view. (B) Top view of the subunit of E2s dimer showing the cavity. β-strands and random coils/turns are depicted in red and green respectively. N- and C-termini are labeled. The dimerization interface and groove region are labeled. (C) The E2s dimer. Subunit A is shown in yellow, subunit B in red. Dimeric interface residues from both subunits are shown in ball-and-stick representation. Notably, the asymmetric unit consists of one subunit of the dimer. This dimer is generated by crystallographic symmetry. These figures were prepared by using Molscript and Raster3D [32],[33]. (D) Close-up view of the dimer interface. Key residues involved in the dimerization are labeled. This figure was prepared using PyMol [34].
Table 1.
Data Collection and Refinement Statistics.
Figure 2.
Dimerization of E2s in solution was investigated by analytical ultracentrifugation (AUC).
(A) Sedimentation velocity experiment shows that E2s behaves as a single globular species having the sedimentation coefficient of 2.55S and a hydrated friction ratio of 1.30. (B) Sedimentation equilibrium experiment indicates that E2s mainly exists as a dimer with M.W. 30,651±421 Da. The dissociation constant of the E2s dimer, Kd was fitted as 397±283 nM using the self-association model.
Figure 3.
Stereo view of the electron density map.
Simulated annealing Fo-Fc omit map of the C-terminal region of E2s, which is crucial for the dimerization. Residues Val600, Leu601 and all atoms within 2.0 Å were omitted prior to refinement. The map contoured at a level of 3σ. This figure was prepared using PyMol [34].
Figure 4.
Structural comparison of E2s with P2 domains.
(A) Side-by-side ribbon diagram of HEV-E2s, the SMSV-P2 domain (pdb code 2gh8) and the rNV-P2 domains (Pdb code 1ihm). β-strands and α-helices are numbered. N- and C-termini are also labeled. These figures were prepared using Molscript and Raster3D [32],[33]. (B) Topology diagrams of HEV-E2s, the SMSV-P2 domain and the rNV-P2 domain. β-strands, α-helices and connecting loops are represented by red arrows, blue cylinders and green lines respectively.
Figure 5.
Mutational studies on the dimer interface.
(A) The schematic representation of wild-type E2 and eleven point mutations targeting the dimer interface region. Secondary structural elements are shown for the E2s region. The mutated residues located on the β-strands and coils are shown in red and black, respectively. (B) These mutants and wild-type E2 were subjected to non-reducing SDS-PAGE and Western Blotting with the neutralizing mAb 8C11 and 8H3 to study the effects of these mutations on dimerization and neutralization, respectively. [+] denotes dimerization or reactivity with 8C11 or 8H3, [−] denotes loss of the respective property. Note that both the capacity to form dimers and the reactivity with mAb 8C11 and 8H3 were abolished simultaneously in six of these mutants: Y557A, T564A, V598E, A599E, L601E and A602E.
Figure 6.
Mapping of site-directed mutation on E2s.
A transparent surface representation of the subunit of the E2s dimer is shown at two different orientations. (A) Shows the dimerization mutants and (B) shows the groove region mutants. Figure 6(B) is 180° rotated with respected to Figure 6(A). Further the view of Figure 6(A) is related to the view of Figure 1(A) with an anticlockwise rotation of 90°. All mutated residues are shown in ball and stick model. Residues playing roles in the E2s dimerization are shown in blue. Residues in the groove region that were mutated to study the reactivity of mAb are shown in magenta. This figure was prepared using PyMol [34].
Figure 7.
Mutational studies on the groove region.
(A) The schematic representation of wild-type E2 and nine point mutations targeting the solvent-accessible residues near the groove region. (B) The wild type E2 and its mutants were subjected to non-reducing SDS-PAGE and Western Blotting with the HEV-neutralizing antibody 8C11 or 8H3. In this figure the lanes with H indicate samples in the reduced condition (i.e. these samples were heated up to 100°C for 3 minutes). These samples were mainly resolved as monomers. The lanes with N indicate samples in the non-reducing condition (i.e. these samples with 0.1% SDS, no BME and were not heated). These samples were resolved mainly as dimers. All nine mutants remained as dimers. Western Blotting showed that the dimeric E2 wild type and eight mutants were reactive with mAb 8C11. Of these, only E479A, Y485A, I529A, K534A and D496A abolished the 8H3 reactivity. Interestingly, mutant D496A abolished the HEV neutralizing antibodies 8C11 and 8H3 reactivity while maintaining the dimeric arrangement.