NEW STRUCTURAL CLASS OF ANTIGEN BINDING SITE IN DROMEDARY SINGLE-DOMAIN VHH IN COMPLEX WITH BOVINE RNASE A

Decanniere K., Desmyter A., Lauwereys, M., Muyldermans, S. & Wyns, L.

Laboratorium voor Ultrastruktuur Vrije Universiteit Brussel Vlaams Interuniversitair Instituut voor Biotechnologie Paardenstraat 65 B-1640 Brussel

Keywords: single-domain antibody, canonical structure

The variable domains of camelid heavy-chain antibodies (VHH) constitute the smallest intact antigen binding site. A crystal structure of the camelid VHH antibody cAb-RN05 in complex with its antigen RNase A has been solved at 2.8 A. The binding surface of the antibody is formed by the hypervariable loop regions H1 and H3 only, with a total binding surface of 570 A2. The overall b-sheet framework of the cAb-RN05 molecule is very similar to known conventional VH structures, except at the "former VL interaction site". At this site, the molecule has a number of substitutions expected for camel single-domain antibodies.

The hypervariable loop H1 of cAb-RN05 has a unique sequence containing three Tyrosine residues at positions 27, 29 and 31. The loop does not adopt any of the known canonical H1 structures.

The H3-loop of cAb-RN05 consists of only 11 amino acids. It folds back over the former VL-interaction surface to cover a number of hydrophobic residues otherwise involved in VL-interactions. The loop is also involved in antigen binding, and folding back the H3-loop enlarges the antigen binding surface.

Previously, the structure of the camelid single-domain antibody cAb-Lys3 bound to its antigen lysozyme was solved in our lab. This structure was characterized by a long H3-loop capable of binding into the active site cleft of lysozyme. The H3-loop in cAb-Lys3 is stabilized by a disulfide bond, and the antibody is a competitive inhibitor of the enzyme. In the cAb-RN05 structure, the H3-loop is to short to form a protruding loop, and the antibody does not bind into the active site cleft. Therefore, cAb-RN05 is not an inhibitor of RNase A. The structure of cAb-RN05 bound to its antigen RNase A shows the strategies that can be used to cover the former VL-interaction and provide a sufficiently large antigen binding surface at the same time with a short H3-loop. 

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