Experimental and Computational Studies on Domain-Swapped Structure Stabilization of an Antibody Light Chain by...

Area of Science:

• Biochemistry
• Structural Biology
• Immunology

Background:

• Antibody-based drugs require efficient design platforms.
• Three-dimensional domain swapping (3D-DS) can occur in antibody variable regions.
• The variable region of antibody light chain #4C214A is susceptible to 3D-DS, forming dimers and tetramers.

Purpose of the Study:

• To stabilize the three-dimensional domain-swapped dimer structure in antibody light chain #4C214A.
• To investigate methods for controlling the dimerization and dissociation of antibody variable regions.

Main Methods:

• Engineered antibody #4C214A by introducing cysteine residues at positions Val2 and Thr97 to form a disulfide bond (#4 V2C/T97C/C214A).
• Assessed tetramer stability at low protein concentration (6 μM).
• Investigated tetramer-to-monomer conversion upon disulfide bond reduction.
• Performed two-dimensional free energy profile analysis using molecular dynamics simulations for tetramerization.

Main Results:

• The engineered #4 V2C/T97C/C214A tetramer remained stable at low protein concentrations.
• Disulfide bond reduction led to the conversion of some tetramers to monomers.
• Molecular dynamics simulations provided insights into the tetramerization process.

Conclusions:

• Disulfide bond introduction is an effective strategy for controlling the dimerization/dissociation of antibody variable regions via 3D-DS.
• This approach offers potential for improving the efficiency and stability of antibody-based drugs.