|Title||A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops.|
|Publication Type||Journal Article|
|Year of Publication||1999|
|Authors||Decanniere, K., A. Desmyter, M. Lauwereys, M. A. Ghahroudi, S. Muyldermans, and L. Wyns|
|Date Published||1999 Apr 15|
|Keywords||Amino Acid Sequence, Animals, Antibody Affinity, Antibody Specificity, Antigen-Antibody Complex, Antigen-Antibody Reactions, Binding Sites, Antibody, Camels, Cattle, Crystallography, X-Ray, Humans, Immunoglobulin Heavy Chains, Mice, Models, Molecular, Molecular Sequence Data, Pancreas, Protein Conformation, Ribonuclease, Pancreatic, Software, Species Specificity|
BACKGROUND: Camelid serum contains a large fraction of functional heavy-chain antibodies - homodimers of heavy chains without light chains. The variable domains of these heavy-chain antibodies (VHH) have a long complementarity 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 inhibitor. The structure of the cAb-RN05-RNase A complex has been solved at 2.8 A. The VHH scaffold architecture is close to that of a human VH (variable heavy chain). The structure of the antigen-binding hypervariable 1 loop (H1) of both cAb-RN05 and cAb-Lys3 differ from the known canonical structures; in addition these H1 loops resemble each other. The CDR3 provides an antigen-binding surface and shields the face of the domain that interacts with VL in conventional antibodies.CONCLUSIONS: VHHs adopt the common immunoglobulin fold of variable domains, but the antigen-binding loops deviate from the predicted canonical structure. We define a new canonical structure for the H1 loop of immunoglobulins, with cAb-RN05 and cAb-Lys3 as reference structures. This new loop structure might also occur in human or mouse VH domains. Surprisingly, only two loops are involved in antigen recognition; the CDR2 does not participate. Nevertheless, the antigen binding occurs with nanomolar affinities because of a preferential usage of mainchain atoms for antigen interaction.