The conformation of an antibody fragment is modified by an unusual cysteine VL87 without interfering with the disulfide bond formation

AGUILAR, MARÍA FERNANDA; ATTALLAH, CAROLINA; A. SERGIO GARAY; FERNANDO E. HERRERA; ETCHEVERRIGARAY, MARINA; OGGERO, MARCOS; DANIEL E. RODRIGUES

Buenos Aires

Congreso; Reunión conjunta de Sociedades de Biociencias, XLVI Reunión anual de la Sociedad Argentina de Biofísica (SAB); 2017

Sociedad Argentina de Biofísica

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 antirecombinant human follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv derived from a monoclonal antibody (mAb) that naturally contains 5 Cys residues: VH22 and VH92 in the variable heavy chain (VH) and VL23, VL87 and VL88 in the variable light chain (VL). The influence of Cys at positions VL87 and VL88 was studied by considering the wild type fragment and mutant variants: C88S, C87S, and C87Y. The analysis was carried out using antigenbinding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations (MD) and docking. We found that Cys VL87 affected theantibody fragment stability and its ability to bind the antigen without interfering with the disulfide bond formation. In the C88S mutant, the CysVL87 was not able to replace the Cys VL88 role and the antibody loss its ability to bind rhFSH. Nevertheless, C87S and C87Y mutants increased the ability of the antibody to bind rhFSH. From the molecular dynamics study we found that the mutant C87Y caused a conformational change in the VH-CDR3 loop, increased flexibility of the V H -CDR2 and V H CDR3 loops and a relative rotation of the VH and VL domains. These changes also comprised the increment in the total number of interchain hydrogen bonds that brings more stability to the multidomain structure. The docking simulations of the wild type scFv, C87S and C87Y mutants on hFSH proposed that the last variant has the larger probability to bind the target. The docking studies also suggested that the paratope loops with larger interaction with the epitope are V H CDR2, V H CDR3 and therefore are prone to be affected by the conformations or mobility of these zones.