Antigen binding and solubility effects upon the veneering of a camel VHH in framework-2 to mimic a VH.

TitleAntigen binding and solubility effects upon the veneering of a camel VHH in framework-2 to mimic a VH.
Publication TypeJournal Article
Year of Publication2005
AuthorsConrath, K., C. Vincke, B. Stijlemans, J. Schymkowitz, K. Decanniere, L. Wyns, S. Muyldermans, and R. Loris
JournalJ Mol Biol
Volume350
Issue1
Pagination112-25
Date Published2005 Jul 1
ISSN0022-2836
KeywordsAmino Acid Sequence, Animals, Antigens, Camels, Chromatography, Gel, Crystallography, X-Ray, Dimerization, Immunoglobulin Variable Region, Models, Molecular, Molecular Sequence Data, Protein Structure, Quaternary, Protein Structure, Tertiary, Sequence Alignment, Solubility
Abstract

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.

DOI10.1016/j.jmb.2005.04.050
Alternate JournalJ. Mol. Biol.
PubMed ID15913651