Role of binding site residues in the sequence-specific recognition ofssDNA by an autoantibody

Defining the structural and thermodynamic basis of protein-nucleic acid recognition is of fundamental importance to understanding cellular regulation. Extensive research has been conducted on protein·dsDNA and protein·RNA systems to elucidate the factors that regulate both binding and specificity. While a large number of proteins that bind sequence-specifically to ssDNA have been reported, a systematic evaluation of sequence-specific recognition has yet to be described.; Antibodies that bind nucleic acids provide a unique model system to characterize nucleic acid-binding proteins. Antibodies all possess a conserved structural framework with variations only in the N-terminal binding site and can be used to study how small changes in protein sequence affects ligand recognition. Antibodies that bind DNA are of interest from a disease perspective because they are produced in patients with the autoimmune disorders. Deposition of immune complexes within kidney tissue can mediate an inflammatory reaction, which can be fatal. Hence, defining the molecular basis of DNA recognition by lupus autoantibodies could provide insight into the pathogenic nature of anti-DNA as well as recognition of DNA in general.; Previous studies have demonstrated that anti-DNA antibody 11F8 localizes to sites of kidney injury through binding of DNA adherent to the glomerular basement membrane. 11F8 binds sequence-specifically to a ssDNA ligand, that is related to DNA antigens found in serum of lupus patients. For 11F8, both the thermodynamic and kinetic basis for specific and non-specific binding has been determined. However, the role of protein residues that mediate binding has not been investigated.; This thesis presents the mutagenesis and binding experiments designed to elucidate the protein residues that mediate recognition of ssDNA. Results reveal six residues of 11F8 account for ca. 80% of the binding free energy. Germline-encoded hydrophobic residues provide for non-specific recognition of thymine bases. Sequence-specific recognition is controlled by the interaction of a non-conserved arginine residue with the loop closing base pair of the stem-loop consensus sequence. The salient features of sequence-specific recognition of ssDNA by 11F8 employ aspects of the paradigms invoked to describe protein·dsDNA, protein·RNA, and antibody-antigen binding and suggest recognition of ssDNA is not identical to any one system.