Mutational analysis of a sequence-specific ssDNA binding lupus autoantibody

Area of Science:

• Immunology
• Structural Biology
• Biochemistry

Background:

• 11F8 is a murine monoclonal autoantibody that recognizes single-stranded DNA (ssDNA).
• Previous studies established the thermodynamic and kinetic basis for 11F8's sequence-specific ssDNA binding.
• Understanding the molecular basis of ssDNA recognition is crucial for autoimmune disease research.

Purpose of the Study:

• To identify specific amino acid residues in 11F8 responsible for sequence-specific, non-cognate, and non-specific ssDNA recognition.
• To determine the contribution of these residues to the overall binding thermodynamics.
• To compare the binding mechanism of 11F8 to other nucleic acid-binding proteins.

Main Methods:

• Site-directed mutagenesis of an 11F8 single-chain construct.
• Analysis of binding free energy contributions from individual residues.
• Comparison of 11F8 primary sequence with clonally related antibodies.

Main Results:

• Six residues in the complementarity determining regions (CDRs) account for approximately 80% of the binding free energy.
• Nonspecific binding of thymine nucleobases is mediated by germline-encoded aromatic and hydrophobic side chains.
• Sequence-specific recognition involves a heavy chain tyrosine and a somatically mutated arginine residue.
• The binding mechanism of 11F8 shares similarities with RNA-binding proteins like U1A.

Conclusions:

• The identified residues are critical for the sequence-specific recognition of ssDNA by 11F8.
• Somatic mutations conferring sequence specificity may differentiate pathogenic anti-DNA autoantibodies from benign ones.
• The study provides insights into the molecular mechanisms underlying autoimmune responses in lupus.