Even when antibodies have high affinity for antigens on a larger structure, such as an intact viral particle, antibody binding may be prevented by their particular arrangement. For example, the intact West Nile virion is built from an icosahedral scaffold that has 90 homodimers of a membrane-anchored envelope glycoprotein, E, which has three domains, DI, DII, and DIII. The DIII domain has four polypeptide loops that protrude outward from the viral particle. A neutralizing antibody against West Nile virus, E16, recognizes these loops of DIII, as shown in Fig. 4.11. In theory, there should be 180 possible antigen-binding sites for the E16 antibody on the West Nile viral particle. However, a combination of crystallographic and electron micrographic studies show that even with an excess of the E16 Fab fragment, only about 120 of the total 180 DIII domains of E are able to bind E16 Fab fragment (see Fig. 4.11).
Fig. 4.10 The complex of lysozyme with the antibody D1.3. Top panel: The interaction of the fab fragment of D1.3 with hen egg-white lysozyme (heL) is shown. heL is depicted in yellow, the heavy chain (Vh) in turquoise, and the light chain (VL) in green. Bottom panel: A glutamine residue (gln121) that protrudes from heL (yellow) extends its side chain (shown in red) between the VL (green) and Vh (turquoise) domains of the antigen-binding site and makes hydrogen bonds with the hydroxyl group (red dots) of the indicated amino acids of both domains. These hydrogen bonds are important to the antigen–antibody binding. courtesy of R. Mariuzza and R.J. Poljak.
Fig. 4.11 Steric hindrance occludes the binding of antibody to native antigen in the intact West Nile viral particle. Top panel: the monoclonal antibody e16 recognizes Diii, one of the three structural domains in the West nile virus glycoprotein e. Shown is a crystal structure of the e16 fab bound to the Diii epitope. Bottom left panel: a computer model was used to dock e16 fab to the mature West nile virion. e16 fabs were able to bind 120 of the 180 Diii epitopes. Sixty of the five-fold clustered Diii epitopes are sterically hindered by the binding of fab to four nearby Diii epitopes. An example of an occluded epitope is the blue area indicated by the arrow. Bottom right panel: cryogenic electron microscopic reconstruction of saturating e16 fab bound to West nile virion confirmed the predicted steric hindrance. The vertices of the triangle shown in the figure indicate the icosahedral symmetry axes.
This appears to result from steric hindrance, with the presence of one Fab blocking the ability of another Fab to bind to some nearby E protein sites. Presumably, such steric hindrance would become more severe with intact antibody than is evident with the smaller Fab fragment. This study also showed that the Fab bound to the DIII region using only one of its antigen-binding arms, indicating that antibodies may not always bind to antigens with both antigen-binding sites, depending on the orientation of the antigens being recognized. These constraints will impact the ability of antibodies to neutralize their targets.
4-10 Some species generate antibodies with alternative structures.