A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops

Background: Camelid serum contains a large fraction of functional heavy cha in antibodies - homodimers of heavy chains without light chains. The variab le domains of these heavy-chain antibodies (VHH) have a long complementarit y determining region 3 (CDR3) loop that compensates for the absence of the antigen-binding loops of the variable light chains (VL). In the case of the VHH fragment cAb-Lys3, part of the 24 amino acid long CDR3 loop protrudes from the antigen-binding surface and inserts into the active-site cleft of its antigen, rendering cAb-Lys3 a competitive enzyme inhibitor. Results: A dromedary VHH with specificity for bovine RNase A, cAb-RN05, has a short CDR3 loop of 12 amino acids and is not a competitive enzyme inhibi tor. The structure of the cAb-RN05-RNase A complex has been solved at 2.8 A ngstrom. The VHH scaffold architecture is close to that of a human VH (vari able heavy chain). The structure of the antigen-binding hypervariable 1 loo p (H1) of both cAb-RN05 and cAb-Lys3 differ from the known canonical struct ures; in addition these H1 loops resemble each other. The CDR3 provides an antigen-binding surface and shields the face of the domain that interacts w ith VL in conventional antibodies. Conclusions: VHHs adopt the common immunoglobulin fold of variable domains, but the antigen-binding loops deviate from the predicted canonical structu re. We define a new canonical structure for the H1 loop of immunoglobulins, with cAb-RN05 and cAb-Lys3 as reference structures. This new loop structur e might also occur in human or mouse VH domains. Surprisingly, only two loo ps are involved in antigen recognition; the CDR2 does not participate. Neve rtheless, the antigen binding occurs with nanomolar affinities because of a preferential usage of mainchain atoms for antigen interaction.