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

摘要: Heavy chain only antibodies of camelids bind their antigens with a single domain, the VHH, which acquired adaptations relative to classical VHs function in absence VL partner. Additional CDR loop conformations, outside canonical structures VHs, broaden repertoire antigen-binding site. The combined effects part CDR3 that folds over "former" binding site and framework-2 mutations more hydrophilic amino acids, enhance solubility VHH domains prevent pairing. cAbAn33, domain specific for carbohydrate moiety variant surface glycoprotein trypanosomes, has short does not cover former as well VH-specific Trp47 instead VHH-specific Gly47. Resurfacing its region (mutations Tyr37Val, Glu44Gly Arg45Leu) mimic human VH restores capacity. In solution, humanised behaves soluble, monomeric entity, albeit reduced thermodynamic stability affinity antigen. Comparison crystal cAbAn33 derivative reveals steric hindrance exerted by residues Tyr37 Arg45 pairing, whereas Glu44 are key elements avoid insolubility domain.