The structural basis for DNA binding by an anti-DNA autoantibody

We have used single and multiple site-directed mutagenesis, and molecular m odeling, to identify critical residues in the DNA binding site of MAb 2C10, an IgG anti-dsDNA autoantibody from an MRL/lpr lupus mouse. Simultaneous r eplacement of four Arg residues in the CDR3H abolished binding activity. Wi th one exception, replacement of any one of these Arg residues reduced the activity to 20-50% of the unmutated scFv activity. Arg to Asp replacements had a slightly greater effect than Arg to Ala replacements. In the one exce ptional case, replacement of Arg99 with Ala actually increased DNA binding five-fold and replacement by Asp had little effect. Mutation of Phe32 and A sn35 to Ala in CDR1H decreased DNA binding, whereas replacement of Arg31 wi th Ala had negligible effect. Ala substitution of any one of a cluster of A sp residues in CDR1L increased DNA binding three to six-fold, confirming pr evious findings that the L-chain of MAb 2C10 is not favorable for DNA bindi ng. The L-chain does participate in shaping the selectivity of antigen bind ing, and mutation of CDR3L residue Asp92 or Asn93 caused a decrease in DNA binding activity. Directed mutagenesis, consistent with a molecular model, indicates that: several CDR amino acids contribute to DNA binding, without one residue dominating; both VH and VL CDR3 domains contribute to specifici ty of binding whereas the CDR1L hinders DNA binding. The results suggest a significant role for electrostatics in the interaction of DNA with MAb 2C10 . (C) 1999 Elsevier Science Ltd. All rights reserved.