Experimental and Computational Studies on Domain-Swapped\nStructure Stabilization of an Antibody Light Chain by Disulfide Bond\nIntroduction

Development of different platforms would be useful for designing\nfunctional antibodies to improve the efficiency of antibody-based\ndrugs. Three-dimensional domain swapping (3D-DS) may occur in the\nvariable region of antibody light chain #4C214A, and a pair of domain-swapped\ndimers may interact with each other to form a tetramer. In this study,\nto stabilize the 3D-DS dimer structure in #4C214A, Val2 in strand\nA (swapping region) and Thr97 in strand G were replaced with Cys residues,\ngenerating #4 V2C/T97C/C214A with a Cys2–Cys97 disulfide bond\nthat cross-links strands A and G of different protomers. The #4 V2C/T97C/C214A\ntetramer did not dissociate into monomers at low protein concentration\n(6 μM); however, some of the tetramers were converted to monomers\nby disulfide bond reduction. Two-dimensional free energy profile analysis\nfor the tetramerization of two 3D-DS dimers was performed by molecular\ndynamics simulation. These results show that disulfide bond introduction\nis useful for controlling the dimerization/dissociation of the variable\nregion through 3D-DS.