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Engineered antibody paratopes with limited sequence diversity permit assessment of the roles played by different amino acid side chains in creating the high-affinity, high-specificity interactions characteristic of antibodies. We describe a paratope raised against the human ErbB family member HER2, using a binary diversity tryptophan/serine library displayed on phage. Fab37 binds to the extracellular domain of HER2 with sub-nanomolar affinity. An X-ray structure at 3.2 A resolution reveals a contact paratope composed almost entirely of tryptophan and serine residues. Mutagenesis experiments reveal which of these side chains are more important for direct antigen interactions and which are more important for conformational flexibility. The crystal lattice contains an unprecedented trimeric arrangement of HER2 closely related to previously observed homodimers of the related epidermal growth factor receptor.

We tested the functional capacity of the natural amino acids for molecular recognition in a minimalist background of binary Tyr/Ser diversity. In phage-displayed synthetic antibody libraries, we replaced either Tyr or Ser with other residues. We find that Tyr is optimal for mediating contacts that contribute favourably to both affinity and specificity, but it can be replaced by Trp, which contributes favourably to affinity but is detrimental to specificity. Arg exhibited a limited capacity for mediating molecular recognition but was less effective than either Tyr or Trp, and moreover, was the major contributor to non-specific interactions. Nine other residue types (Phe, Leu, Ile, Asn, Thr, Pro, Cys, Ala, and Gly) were found to be ineffective as replacements for Tyr. By replacing Ser with Gly or Ala, we found that Gly is as effective as Ser for providing conformational flexibility that allows bulky Tyr residues to achieve optimal binding contacts, while Ala is less effective but still functional in this capacity. For some antigens, high affinity antibodies could be derived using only Tyr/Ser/Gly diversity, but for others, additional chemical diversity was required to achieve high affinity. Our results establish a minimal benchmark for the generation of synthetic antigen-binding sites with affinities comparable to those of natural antibodies. Moreover, our findings illuminate the fundamental principles underlying protein-protein interactions and provide valuable guidelines for engineering synthetic binding proteins with functions beyond the scope of natural proteins.