Coevolution of antibody stability and Vκ CDR-L3 canonical structure.
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.
Luo
Jinquan
J
Centocor Research and Development, Inc., Radnor, PA 19087, USA. jluo@its.jnj.com
Obmolova
Galina
G
Huang
Alice
A
Strake
Brandy
B
Teplyakov
Alexey
A
Malia
Thomas
T
Muzammil
Salman
S
Zhao
Yonghong
Y
Gilliland
Gary L
GL
Feng
Yiqing
Y
D000911:Antibodies, Monoclonal / Q000235:genetics*; D022801:Complementarity Determining Regions / Q000235:genetics*; D019020:Directed Molecular Evolution; D018095:Germ-Line Mutation; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D011489:Protein Denaturation; D015202:Protein Engineering; D055550:Protein Stability; D044366:Transition Temperature
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains