Tyrosine Plays a Dominant Functional Role in the Paratope of a Synthetic Antibody Derived from a Four Amino Acid Code

The antigen-binding fragment Fab-YADS2 recognizes vascular endothelial growth factor (VEGF) and was derived from a library with chemical diversity restricted to only four amino acids (Tyr, Ser, Ala and Asp). The structure of the Fab:antigen complex revealed that the structural paratope is dominated by Tyr side-chains. Isothermal titration calorimetry and cell-based assays show that restricted chemical diversity does not limit the affinity or specificity of Fab-YADS2, which behaves in a manner comparable to natural antibodies. Mutagenesis experiments reveal that the functional paratope is dominated by Tyr, which represents 11 of the 15 functionally important residues. However, mutagenesis experiments also indicate that substitution of any of these tyrosine residues by Phe does not significantly affect binding to VEGF. Furthermore, saturation mutagenesis shows that replacement of three functionally important tyrosine residues by combinations of other hydrophobic residues is not only tolerated, but can actually improve affinity. The results support a model for naïve antigen recognition in which large Tyr side-chains establish binding contacts with antigen, and small Ser and Ala side-chains serve as auxiliaries that help to position Tyr in favorable binding conformations. While Tyr may not be optimal for any particular antigen contact, it is nonetheless capable of mediating favorable interactions with a diverse array of surfaces. Furthermore, the side-chain hydroxyl group makes Tyr significantly more hydrophilic than Phe and other hydrophobic amino acids. Increased hydrophilicity may reduce non-specific binding in the unbound state, and this may be critical for a naïve repertoire that is exposed to a diverse range of potential antigenic surfaces. The results show that the chemical nature of Tyr endows the amino acid with a privileged role in antigen recognition, and this likely explains the high abundance of Tyr in natural antigen-binding sites.