Antigen specificity and high affinity binding provided by one single loop of a camel single-domain antibody

Detailed knowledge on antibody-antigen recognition is scarce given the unli mited antibody specificities of which only few have been investigated at an atomic level. We report the crystal structures of an antibody fragment der ived from a camel heavy chain antibody against carbonic anhydrase, free and in complex with antigen. Surprisingly, this single-domain antibody interac ts with nanomolar affinity with the antigen through its third hypervariable loop (19 amino acids long), providing a flat interacting surface of 620 An gstrom (2). For the first time, a single-domain antibody is observed with i ts first hypervariable loop adopting a type-1 canonical structure. The seco nd hypervariable loop, of unique size due to a somatic mutation, reveals a regular p-turn, The third hypervariable loop covers the remaining hypervari able loops and the side of the domain that normally interacts with the vari able domain of the light chain, Specific amino acid substitutions and reori ented side chains reshape this side of the domain and increase its hydrophi licity, Of interest is the substitution of the conserved Trp-103 by Arg bec ause it opens new perspectives to 'humanize' a camel variable domain of hea vy chain of heavy chain antibody (VHH) or to 'camelize' a human or a mouse variable domain of heavy chain of conventional antibody (VH).