HHHHHH HH NH OOC3 N H O C ˜–˜–˜+˜–˜–˜+˜+˜–˜˜–˜–˜–˜–˜–˜––˜+˜+H H O H HO HH O Na+H H O Noncovalent forces Origin Electrostatic forces Hydrogen bonds Attraction between opposite charges Hydrogen shared between electronegative atoms (N, O) Van der Waals forces Hydrophobic forces Cation-pi interaction Fluctuations in electron clouds around molecules polarize neighboring atoms oppositely Hydrophobic groups interact unfavorably with water and tend to pack together to exclude water molecules. The attraction also involves van der Waals forces Non-covalent interaction between a cation and an electron cloud of a nearby aromatic group H H
Fig. 4.9 The noncovalent forces that hold together the antigen:antibody complex. Partial charges found in electric dipoles are shown as δ+ or δ–. electrostatic forces diminish as the inverse square of the distance separating the charges, whereas van der Waals forces, which are more numerous in most antigen–antibody contacts, fall off as the sixth power of the separation and therefore operate only over very short ranges. covalent bonds never occur between antigens and naturally produced antibodies.
VL VH D1.3 Gln121 in their antigen-binding sites. These amino acids participate mainly in van der Waals and hydrophobic interactions, and sometimes in hydrogen bonds and pi-cation interactions. Tyrosine, for example, can take part in both hydrogen bonding and hydrophobic interactions; it is therefore particularly suitable for providing diversity in antigen recognition and is overrepresented in antigen-binding sites. In general, the hydrophobic and van der Waals forces operate over very short ranges and serve to pull together two surfaces that are complementary in shape: hills on one surface must fit into valleys on the other for good binding to occur. In contrast, electrostatic interactions between charged side chains, and hydrogen bonds bridging oxygen and/or nitrogen atoms, accommodate more specific chemical interactions while strengthening the interaction overall. The side chains of aromatic amino acids such as tyrosine can interact noncovalently through their pi-electron system with nearby cations, including nitrogen-containing side chains that may be in a protonated cationic state.
4-9 Antibody interaction with intact antigens is influenced by steric constraints.
An example of an antibody–antigen interaction involving a specific amino acid in the antigen can be seen in the complex of hen egg-white lysozyme with the antibody D1.3 (Fig. 4.10). In this structure, strong hydrogen bonds are formed between the antibody and a particular glutamine in the lysozyme molecule that protrudes between the VH and VL domains. Lysozymes from partridge and turkey have another amino acid in place of the glutamine and do not bind to this antibody. In the high-affinity complex of hen egg-white lysozyme with another antibody, HyHel5 (see Fig. 4.8c), two salt bridges between two basic arginines on the surface of the lysozyme interact with two glutamic acids, one each from the VH CDR1 and CDR2 loops. Lysozymes that lack one of the two arginine residues show a 1000-fold decrease in affinity for HyHel5. Overall surface complementarity must have an important role in antigen–antibody interactions, but in most antibodies that have been studied at this level of detail, only a few residues make a major contribution to the binding energy and hence to the final specificity of the antibody. Although many antibodies naturally bind their ligands with high affinity, in the nanomolar range, genetic engineering by site-directed mutagenesis can tailor an antibody to bind even more strongly to its epitope.