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

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Abstract

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 shod 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 Å. 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.

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Decanniere, K., Desmyter, A., Lauwereys, M., Ghahroudi, M. A., Muyldermans, S., & Wyns, L. (1999). A single-domain antibody fragment in complex with RNase A: Non-canonical loop structures and nanomolar affinity using two CDR loops. Structure, 7(4), 361–370. https://doi.org/10.1016/S0969-2126(99)80049-5

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