An unusual cysteine VL87 affects the antibody fragment conformations without interfering with the disulfide bond formation

Abstract

The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: V_H22 and V_H92 in the variable heavy chain (V_H) and V_L23, V_L87 and V_L88 in the variable light chain (V_L). The influence of two unusual contiguous Cys at positions V_L87 and V_L88 was studied by considering the wild type fragment and mutant variants: V_L-C88S, V_L-C87S, V_L-C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that V_L-C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the V_L-C87 by a usual residue at this position like Tyr caused distant structural changes at the V_H region that confers a higher mobility to the V_H-CDR2 and V_H-CDR3 loops improving the scFv binding to the antigen.

Keywords: Cysteine contiguous residues; Molecular modeling; Mutating cysteine; Single-chain variable fragment.