Mechanistic Insights into How the Single Point Mutation Change the Autoantibody Repertoire

Area of Science:

• Molecular Biology
• Immunology
• Biochemistry

Background:

• Autoantibodies can lose DNA binding ability due to single point mutations, such as F33 to Y in heavy chain complementarity-determining region 1 (H-CDR1).
• The precise molecular mechanisms underlying this loss of function remain incompletely understood.

Purpose of the Study:

• To elucidate the molecular mechanisms by which the F33 to Y mutation in H-CDR1 causes anti-DNA antibodies to lose their DNA binding capability.
• To investigate the contributions of different antibody regions and interaction types to the binding of single-strand DNA (ssDNA).

Main Methods:

• Computational analysis to assess interaction forces (electrostatic, hydrogen bonding, pi-pi stacking).
• Site-directed mutagenesis of key residues (F33H, W98H, Y95L) in anti-DNA antibodies.
• Expression and purification of wildtype and mutant antibodies in CHO cells.
• Enzyme-linked immunosorbent assay (ELISA) to evaluate ssDNA binding affinity.

Main Results:

• Electrostatic forces are not the primary drivers of anti-DNA antibody-ssDNA interaction; H-CDR2 significantly contributes to binding, even more than H-CDR1.
• The F33Y mutation enhances hydrogen bonding but disrupts essential pi-pi stacking interactions between the antibody and ssDNA.
• Wildtype antibody residues F33H, W98H, and Y95L form stable pi-pi stacking with ssDNA bases, a critical interaction lost in the Y33 mutant.

Conclusions:

• The F33Y mutation abrogates ssDNA binding primarily by disrupting critical pi-pi stacking interactions, not electrostatic forces.
• H-CDR2 plays a more significant role in ssDNA binding than previously recognized.
• Understanding these interactions is crucial for antibody engineering and developing targeted therapies.