An ultralong CDRH2 in HCV neutralizing antibody demonstrates structural plasticity of antibodies against E2 glycoprotein
- Division of Biology and Biological Engineering, California Institute of Technology, United States
- Department of Medicine, Johns Hopkins University School of Medicine, United States
Figures
Figure 1
AR3X includes a 14-residue insertion in CDRH2.
(a) Sequence alignment of a portion of the heavy chain variable region gene sequences of AR3X and the AR3X germline precursor (AR3Xrua) (uppercase letters) and the VH1-69 gene segment (lowercase …
Figure 2 with 2 supplements
The CDRH2 insertion in AR3X is required for maximal binding and broad neutralization.
(a) Heat map showing the binding of AR3X and its variants to a panel of HCV E2ecto proteins. The EC50 value for each E2ecto-mAb combination is shown, with dark red, orange, yellow, or white shading …
Figure 2—figure supplement 1
Binding of AR3X and its variants to a panel E2ecto proteins.
Values shown are means ± s.d. of duplicates. One experiment representative of two independent experiments is shown.
Figure 2—figure supplement 2
Neutralization activities of AR3X and its variants against a panel of genotype 1 HCVpp.
Values shown are means ± s.d. of duplicates.
Figure 3
The shared CDRH3 motif in E2 front layer-specific HCV bNAbs adopts different orientations.
Fab structures in liganded state of AR3A (PDB 6BKB), AR3C (PDB 4MWF), AR3X (this paper), HEPC3 (PDB 6MEI), and HEPC74 (PDB 6MEH). The structures were superimposed on their VH domains. Protein …
Figure 4 with 2 supplements
Details of the AR3X interactions with E2ecto.
(a) Crystal structure of the AR3X-E2ecto complex. E2ecto is shown as a cartoon representation within a transparent surface with N-glycans highlighted as sticks and disulfide bonds shown as yellow …
Figure 4—figure supplement 1
Data collection and refinement statistics for AR3X-E2ecto1b09 complex.
Figure 4—figure supplement 2
Interface residues between AR3X and E2ecto.
Figure 5
A structural plasticity of VH1-69-derived bNAbs with the CDRH3 disulfide motif.
(Top) Surface representations of AR3X-E2 and other bNAb-E2 structures. E2, gray; AR3A-HC, red; AR3A-LC, light red; AR3C-HC, orange; AR3C-LC, yellow; AR3X-HC, green; AR3X-LC, light green; HEPC3-HC, …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo-sapiens) | HEK293-6E | National Research Council of Canada | 11565 | |
| Cell line (Homo-sapiens) | Expi293F | Thermo Fisher Scientific | A14527 | |
| Cell line (Homo-sapiens) | Hep3B2.1–7 | ATCC | HB-8064 | |
| Antibody | Anti-Human IgG-HRP (Goat polyclonal) | SouthernBiotech | 2040–05 | 1:4000 dilution |
| Recombinant DNA reagent | pTT5 mammalian expression vector (used to express IgGs and Fabs) | National Research Council of Canada | N/A | |
| Commercial assay or kit | 1-Step Ultra TMB-ELISA Substrate Solution | Thermo Fisher Scientific | 34028 | |
| Commercial assay or kit | PEGRx HT | Hampton Research | HR2-086 | |
| Commercial assay or kit | PEG/Ion HT | Hampton Research | HR2-139 | |
| Commercial assay or kit | JCSG-plus HT-96 | Molecular Dimensions | MD1-40 | |
| Chemical compound, drug | Kifunensine | Sigma | K1140 | |
| Software, algorithm | Pymol | Schrödinger, LLC | RRID:SCR_000305 | |
| Software, algorithm | Phenix | (Adams et al., 2010) | https://www.phenix-online.org | |
| Software, algorithm | Coot | (Emsley and Cowtan, 2004) | http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | |
| Software, algorithm | PDBePISA | (Krissinel and Henrick, 2007) | http://www.ebi.ac.uk/pdbe/pisa/ | |
| Software, algorithm | abYsis system | http://www.bioinf.org.uk/abysis/ | ||
| Other | Superdex 200 Increase 10/300 GL | GE Healthcare | 17517501 | |
| Other | HisTrap FF column | GE Healthcare | 17531901 | |
| Other | HiTrap Protein A HP column | GE Healthcare | 17040301 | |
| Other | HCV 1b09 strain E1E2 sequence | GenBank | KJ187984.1 |
