NP [BSA-Biotin]

Antibodies with a basic four-chain structure with two binding sites interact with various types of antigens and also act as antigen receptors on B cells. Antibodies bind specifically to antigen through complementary decision region (CDR) loops on variable heavy and light chains (VH and VL). Affinity maturation of antibodies, including somatic hypermutation, is a form of protein evolution in nature. It is well known that affinity maturation improves the binding affinity and specificity of an antibody to its immunogen through somatic hypermutation. A hapten (4-hydroxy-3-nitrophenyl) acetyl (NP) has been used to analyze the structural basis of affinity maturation. Some anti-NP monoclonal antibodies are referred to as affinity-matured typical antibodies; N1G9 and F8 are germline antibodies with Trp33^H and Tyr95^H, B2 is an affinity-matured antibody with Leu33^H and Tyr95^H, and 9T7, C6, and E11 are affinity-matured antibodies with Trp33^H and Gly95^H respectively.

Based on the amino acid residue at position 95 of the heavy chain, which corresponds to the junction of the VH and D segments, anti-NP antibodies can be classified into at least two types: Tyr95-type and Gly95-type. The investigators found that the W33L mutation resulted in an increase in the affinity of the Tyr95-type antibody and a decrease in the affinity of the Gly95-type antibody, which utilizes other mutations to obtain higher affinity, suggesting that affinity maturation of Tyr95-type and Gly95-type anti-NP antibodies occurs through different strategies. In addition to the different strategies employed, Tyr95-type and Gly95-type affinities have different upper affinity limits reached after maturation. The binding constants of germline anti-NP antibodies range from 10⁵-10⁶ M^-1 without considering the case of amino acid 95, while the upper affinity limits after affinity maturation are ∼10⁷ M^-1 for Tyr-95 types and ∼10⁹ M^-1 for Gly95 types.

Several investigators have analyzed the structural properties of anti-NP antibodies during affinity maturation. The change from Trp to Leu at position 33 of the heavy chain by somatic mutation (Trp33^HLeu) is essential for the maturation of the Tyr95^H -type antibody N1G9, which increases the affinity approximately 10-fold. Some researchers have found that the amino acid residue in the 95th position of the heavy chain is critical for improving antigen-binding affinity in the later stages of immunization. Since anti-NP antibodies with Gly95^H are able to obtain higher affinity by affinity maturation, whereas anti-NP antibodies with Tyr95^H are not, it is of interest to compare the crystal structures of Tyr95^H -type N1G9 and Gly95^H -type C6 from the point of view of how anti-NP antibodies with Gly95^H can obtain high affinity at the atomic level.

Figure 1. Close up view of the NP binding sites of C6 and N1G9
(Source: Nishiguchi A, et al. 2019)

(4-hydroxy-3-nitrophenyl)acetyl [BSA-Biotin]