The monoclonal anti-testosterone antibody (3-C4F5) has a relatively high affinity (3 × 108 M −1) with an overall good specificity profile. However, the earlier characterized binding properties have shown that both the affinity and specificity of this antibody must be improved if it is intended for use in clinical immunoassays. In this paper, the crystal structures of the recombinant anti-testosterone (3-C4F5) Fab fragment have been determined in the testosterone-bound and free form at resolutions of 2.60 and 2.72 Å, respectively. The high affinity binding of the (3-C4F5) Fab is mainly determined by shape complementarity between the protein and testosterone. Only one direct hydrogen bond is formed between the hydroxyl group of the testosterone D-ring and the main-chain oxygen of Gly100JH. The testosterone is deeply bound in a hydrophobic pocket, and the close shape complementarity is mainly formed by the third complementarity-determining regions (CDR) of the heavy and light chain. Comparison of the bound structure with the free structure indicates conformational changes in the protein upon testosterone binding. The conformational changes of the side chains of two residues Glu95H and Tyr99H in the CDR-H3 are particularly essential for the binding. Interesting similarities in the binding of different steroids were also observed upon comparison of the available structures of anti-steroid antibodies.
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 2002|
|MoE publication type||A1 Journal article-refereed|