11F8 is a pathogenic monoclonal anti-ssDNA autoantibody isolated from a lupus prone mouse. Previous studies established that 11F8 is sequence-specific and identified the thermodynamic and kinetic basis for the specific recognition of ssDNA, and binding site mutations of a single-chain construct reveal that (Y32)LCDR1, (R31)HCDR1, (W33)HCDR1, (R98)HCDR3, (L97)HCDR3, and (Y100)HCDR3 are responsible for approximately 80% of the binding free energy. Here we evaluate the role of these residues along with a group of basic residues (K62, K64, R24, K52) within the context of the binding mechanism. Binding of 11F8 takes place in two steps. In the first step, the overall positive charge of the antigen binding site attracts the negatively charged DNA to form an encounter complex that is stabilized by two salt bridges and a hydrogen bond. The second step is a slow process in which minor conformational changes occur. During this step, aromatic side chains become desolvated, presumably through stacking interactions involving two thymine bases within the DNA recognition epitope. Although the stability of the complex arises primarily from interactions formed in the second step, sequence specificity results from interactions with residues involved in both steps. These studies also show that the way in which 11F8 achieves high affinity sequence-specific binding is more closely related to RNA binding proteins than those that bind DNA and point to strategies for disrupting DNA binding that could prove to be therapeutically useful.

11F8 is a sequence-specific pathogenic anti-single-stranded (ss)DNA autoantibody isolated from a lupus prone mouse. Site-directed mutagenesis of 11F8 has shown that six binding site residues (R31VH, W33VH, L97VH, R98VH, Y100VH, and Y32VL) contribute 80% of the free energy for complex formation. Mutagenesis results along with intermolecular distances obtained from fluorescence resonance energy transfer were implemented here as restraints to model docking between 11F8 and the sequence-specific ssDNA. The model of the complex suggests that aromatic stacking and two sets of bidentate hydrogen bonds between binding site arginine residues (R31VH and R96VH) and loop nucleotides provide the molecular basis for high affinity and specificity. In part, 11F8 utilizes the same ssDNA binding motif of Y32VL, H91VL, and an aromatic residue in the third complementarity-determining region to recognize thymine-rich sequences as do two anti-ssDNA autoantibodies crystallized in complex with thymine. R31SVH is a dominant somatic mutation found in the J558 germline sequence that is implicated in 11F8 sequence specificity. A model of the mutant R31S11F8.ssDNA complex suggests that different interface contacts occur when serine replaces arginine 31 at the binding site. The modeled contacts between the R31S11F8 mutant and thymine are closely related to those observed in other anti-ssDNA binding antibodies, while we find additional contacts between 11F8 and ssDNA that involve amino acids not utilized by the other antibodies. These data-driven 11F8.ssDNA models provide testable hypotheses concerning interactions that mediate sequence specificity in 11F8 and the effects of somatic mutation on ssDNA recognition.

Autoantibodies that bind DNA are a hallmark of systemic lupus erythematosus. A subset of autoantibody*DNA complexes localize to kidney tissue and lead to damage and even death. 11F8, 9F11, and 15B10 are clonally related anti-DNA autoantibodies isolated from an autoimmune mouse. 11F8 binds ssDNA in a sequence-specific manner and causes tissue damage, while 9F11 and 15B10 bind ssDNA non-specifically and are benign. Among these antibodies, DNA binding properties are mediated by five amino acid differences in primary sequence. Thermodynamic and kinetic parameters associated with recognition of structurally different DNA sequences were determined for each antibody to provide insight toward recognition strategies, and to explore a link between binding properties and disease pathogenesis. A model of 11F8 bound to its high affinity consensus sequence provides a foundation for understanding the differences in thermodynamic and kinetic parameters between the three mAbs. Our data suggest that 11F8 utilizes the proposed ssDNA recognition motif including (Y32)V(L), a hydrogen bonding residue at (91)V(L), and an aromatic residue at the tip of the third heavy chain complementarity determining region. Interestingly, a somatic mutation to arginine at (31)V(H) in 11F8 may afford additional binding site contacts including (R31)V(H), (R96)V(H), and (R98)V(H) that could determine specificity.

11F8 is a sequence-specific monoclonal anti-ssDNA autoantibody isolated from a lupus prone mouse that forms pathogenic complexes with ssDNA, resulting in kidney damage. Prior studies show that specificity is mediated by a somatic mutation from serine at (31)V(H) to arginine. Reversion back to serine in 11F8 resulted in >30-fold decrease in affinity and altered thermodynamic and kinetic parameters for sequence-specific recognition of its cognate ssDNA ligand. Mutagenesis and structural studies suggest that (R31)V(H) contacts ssDNA via a salt bridge and a bidentate hydrogen bond and may further contribute to specificity by altering binding-site conformation. Fluorescence resonance energy transfer experiments were conducted to assess the kinetics of conformational change during 11F8*ssDNA association. The extent of rearrangement between the six complementary determining regions in the 11F8*ssDNA complex with germline serine or somatically mutated arginine at residue 31 of the heavy chain was examined. Our studies show that greater conformational change occurs in five of six complementarity determining regions after the heavy chain germline J558 sequence undergoes mutation to arginine at (31)V(H).