Antigen binding and solubility effects upon the veneering of a camel VHH in framework-2 to mimic a VH.
Heavy chain only antibodies of camelids bind their antigens with a single domain, the VHH, which acquired adaptations relative to classical VHs to function in the absence of a VL partner. Additional CDR loop conformations, outside the canonical loop structures of VHs, broaden the repertoire of the antigen-binding site. The combined effects of part of the CDR3 that folds over the "former" VL binding site and framework-2 mutations to more hydrophilic amino acids, enhance the solubility of VHH domains and prevent VL pairing. cAbAn33, a VHH domain specific for the carbohydrate moiety of the variant surface glycoprotein of trypanosomes, has a short CDR3 loop that does not cover the former VL binding site as well as a VH-specific Trp47 instead of the VHH-specific Gly47. Resurfacing its framework-2 region (mutations Tyr37Val, Glu44Gly and Arg45Leu) to mimic that of a human VH restores the VL binding capacity. In solution, the humanised VHH behaves as a soluble, monomeric entity, albeit with reduced thermodynamic stability and affinity for its antigen. Comparison of the crystal structures of cAbAn33 and its humanised derivative reveals steric hindrance exerted by VHH-specific residues Tyr37 and Arg45 that prevent the VL domain pairing, whereas Glu44 and Arg45 are key elements to avoid insolubility of the domain.
Conrath
Katja
K
Laboratorium voor Cellulaire en Moleculaire Immunologie, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium. kconrath@vub.ac.be
Vincke
Cécile
C
Stijlemans
Benoît
B
Schymkowitz
Joost
J
Decanniere
Klaas
K
Wyns
Lode
L
Muyldermans
Serge
S
Loris
Remy
R
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000737:chemistry* / Q000276:immunology*; D002162:Camelus / Q000276:immunology*; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D019281:Dimerization; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D012995:Solubility
D000941:Antigens; D007135:Immunoglobulin Variable Region