Fig. 8

Structural characterization of the chimeric pentamers and VLPs-Fab immune complexes. a Binding profiles of mAb 4E5 to chimeric mutant and WT VLPs of HPV33, HPV52 and HPV58. Data indicates mAb 4E5 can bind to WT VLPs of HPV33 and chimeric VLPs of H58-33BC and H58-33BC-52HI but fails to bind to WT VLPs of HPV52 and HPV58. The error bars are standard deviation of experiments performed in duplicate. b HPV33 L1 protein was subjected to reducing SDS-PAGE and western blot (WB) analysis with mAb 4E5. mAb 4B3, a linear antibody capable of recognizing HPV L1 protein, was employed as a positive control for the WB experiment. c Affinity constants of the chimeric mutants (H58-33BC and H58-33BC-52HI) and WT VLPs (HPV33, HPV52 and HPV58) against mAb 4E5 were measured by surface plasma resonance (SPR) in a Biacore 3000. The error bars are standard deviation of experiments performed in triplicate. d CryoEM structures of the VLPs-Fab complexes of HPV33:4E5 (left), H58-33BC:4E5 (middle) and H58-33BC-52HI:4E5 (right). Density maps are colored by radius from cyan (230 Å), to yellow (280 Å), to pink (320 Å). Icosahedral 2-, 3- and 5-fold axes are indicated by black symbols. For the purpose of comparability, the cryoEM maps at higher resolution were low-pass filtered to 12.3 Å. e Close-up view of the roadmaps of HPV33:4E5 (left), HPV58-33BC:4E5 (middle) and HPV58-33BC-52HI (right) surfaces, projected by an area surrounding a 5-fold vertex defined with a polar angle θ range of 46° to 58° and a polar angle ϕ range of 68°–86°. Color scheme for surface residues are according to surface loops (BC, amber; DE, orange; EF, yellow; FG, green and HI, Cyan). Blue contour lines were drawn according to the projection of the Fab-4E5 entity with a density greater than 1 and within a radial section of the VLP-4E5 complex ranging from 310 Å to 360 Å. The icosahedral symmetry axes are labeled. The roadmaps were generated by the software RIVEM⁶⁸