Structural Modeling of Sequence Specificity by an Autoantibody against Single-Stranded DNA

Abstract

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.

Gene silencing by cell-penetrating, sequence-selective and nucleic-acid hydrolyzing antibodies

“…In vitro engineering of anti-DNA/RNA antibodies that specifically recognize and hydrolyze specific DNA/RNA antigens has never been reported, although an RNA-binding Fab has been engineered to specifically recognize the targeted RNA tertiary structure (30) and sequence-specific DNA binding antibodies have been raised in vivo from mice (31,32). Our results suggest that proteins with β-sheet structure can be used as a scaffold to engineer sequence-specific ss-DNA/RNA binding proteins, as α-helical zinc-finger protein does in specific-specific ds-DNA-binding proteins (33).…”

Impact of DNA Hairpin Folding Energetics on Antibody–ssDNA Association

“…However, efforts to crystallize 11F8 have thus far proven unsuccessful. 19 A desire to provide structural data relevant to Ab 11F8 was a major motivation for this study. We note that the theoretical models of 11F8 complexed with DNA 12,17,19 are based, in part, on the DNA-1-dT n crystal structures.…”

“…18 Although molecular modeling has been done for this system, crystallization of the 11F8/LIG1-17 complex has been unsuccessful. 19 The focus of the present study is the recombinant anti-single-stranded (ss)DNA antigen-binding fragment (Fab) DNA-1. DNA-1 was isolated from a combinatorial bacteriophage display library of IgG fragments derived from the immunoglobulin repertoire of an autoimmune SLE-like MRL/lpr mouse.…”

Structural Insight into the Tetramerization of an Iterative Ketoreductase SiaM through Aromatic Residues in the Interfaces

“…Frequently, aromatic stacking interactions are found in the interfaces of protein/nucleic acid complexes. The aromatic residues at the binding site form pi-pi stacking with nucleic acids and achieve specific recognition between the binding partners [43], [44]. The aromatic stacking interactions between proteins are relatively rare because the large hydrophobic aromatic residues are seldom located on the surface of proteins.…”