Coevolution of Antibody Stability and Vκ CDR-L3 Canonical Structure

Abstract

Antibodies recognize antigens through six hypervariable loops, five of which have a limited set of conformations known as canonical structures. For κ light chains, the majority of CDR-L3 [the third hypervariable loop of the light chain variable domain (V L)] adopts the type 1 canonical structure (CS1), with a cis-proline at position 95. Here, we present the design and structural studies of the monoclonal antibody mAb15 and related mutants that contained a series of progressively germline mutations only in the heavy chain variable domain (V H) that ultimately led to an increase of more than 11 °C in the melting temperature ( T m) of the antigen-binding fragment (Fab). The all- trans CDR-L3 structure in the wild type is significantly different from any known CDR-L3 canonical structures. In the thermally stable mutants, the L94 L–S95 L peptide bond adopts an energetically unfavorable non-X-proline cis conformation, but the overall CDR-L3 loop converted to CS1. The stabilized V H appears to function as a specific molecular chaperone that facilitated the trans– cis isomerization of S95 L. Thus, it is plausible that proline is the evolutionary choice to maintain overall structure and stability for V L. These results provide new insights into the evolution of CS1 and suggest a potential molecular switch mechanism at position 95 that links CDR-L3 structural diversity and antibody stability and will have implications for antibody engineering.