Potent neutralizing antibodies elicited by dengue vaccine in rhesus macaque target diverse epitopes
Fig 5
Critical residues for binding of the broadly neutralizing antibody d448.
The structural presentation of the critical residues of the epitope on DENV4 domain II. (A) Critical resides (green spheres) are mapped on both monomers of the mature E protein dimer structure (PDB, 1UZG). Domain I is red, domain II yellow, domain III blue and fusion loop cyan. The asymmetric monomer rotated 180 degrees is gray. (B) Sequence alignment of the epitope region on the E protein. Epitope residues D215, P219, Q256, and G266 are conserved among the different serotypes; only one residue M237 in DENV4 is not conserved. (Leucine in the other three serotypes) The NCBI accession numbers of the sequences are ACJ04226 (DENV1), AGS49173 (DENV2), AJA37731 (DENV3) and ACW82884 (DENV4) respectively. (C) Side view of the E:M:M:E heterotetramer. The transmembrane domain of M protein is in magenta, the space-filling model of the ectodomain of M protein is in light blue. Four critical residues (P219, M237, Q256, and G266) of the epitope are located in the hydrophobic core of the E-M interface. One residue (D215), out of the hydrophobic core, contacts the perimembrane helix of the M protein (PDB, 1UZG). (D) Enlarged view shows the contact of D215 with R38 on M protein. The analysis was performed using “Structural Analysis: Distances” of UCSF chimera. (E) Enlargement of the hydrophobic core. The stick model, shown in light blue, presents the first 20 amino acids of M protein (M1-20). The space-filling model of E shows the three hydrophobic pockets on E protein, where the M1-20 binds. The red dotted lines show the boundaries of the pockets. The critical residues of the epitope are indicated. (F) In pocket 2, H209 and T212 on E form a hydrophobic core with His7 on M, and in pocket 3, T19 on M is encompassed by the recess of T206 and H261 on E. The amino acid G266 forms a barrier between pocket 2 and pocket 3, contacting the conserved residues of H7 and H19 on M protein. (G) The amino acids P219, Q256, M237 are on the edge of pocket 1, consistent with L216, L218, and M260 on E protein, these six residues encompass the valine in the position 2 (V2) on M in the pocket 1 center. Atoms are shown in red for O, blue for N, and magenta for S. Molecular graphic images were produced using the UCSF Chimera (http://www.rbvi.ucsf.edu/chimera). (H) Neutralization percentage of the DENV2 reporter virus particles, and its critical amino acid residue substitution mutants. (I) Neutralization percentage of the DENV4 reporter virus particles, and its critical amino acid residue substitution mutants. Antibody d448 was tested at the concentration of 3 μg/ml. The assays were performed in triplicates and data were analyzed using GraphPad Prism.