| Proten•ssDNA interactions are central to many biological processes, and specificity is fundamental to cellular regulation. Antibodies provide a unique model system to study protein•ssDNA interactions because their highly conserved sequence and three-dimensional structure has been mutated to differentiate residues necessary for structural integrity from those involved in antigen binding. Additionally, anti-DNA autoantibodies are a prominent hallmark of the autoimmune disease systemic lupus erythematosus. Through a process including antigen recognition, a subset of anti-DNA localize to the glomerular basement membrane of kidney tissue and initiate an inflammatory response that can result in death. Defining the molecular basis of recognition by lupus antibodies provides insight towards the pathogenic nature of anti-DNA as well as protein•ssDNA recognition.; Previous studies have demonstrated that anti-DNA autoantibody 11F8 binds sequence-specifically to a ssDNA hairpin that bears both sequence and structural similarity to DNA antigens found in serum of lupus patients. Administration of 11F8 induces lupus pathology in normal mice. Site-directed mutagenesis revealed that two amino acids, R31VH and Y100VH, mediate sequence specificity in 11F8; however, the nature of intermolecular contacts with ssDNA could not be determined. 31VH and 100VH differ from the equivalent residues in clonally related non-sequence-specific antibodies, implying that contacts formed with these two residues should provide insight towards the molecular basis of sequence-specificity.; This thesis presents structural and dynamic characterization of 11F8•ssDNA recognition. The tools of mutagenesis, fluorescence resonance energy transfer, stopped-flow kinetics, thermodynamics, and modeling were utilized in concert to describe intermolecular contacts in the sequence-specific complex. Our model suggests that aromatic stacking and pi-cation interactions afford stability, while selectivity arises from two sets of bidentate hydrogen bonds and an aromatic residue in the center of the binding site. The model proposes that a single arginine acquired during antibody maturation affords an extended interface with the ssDNA beyond the contacts accessible for related non-specific anti-ssDNA autoantibodies. Dynamics studies support that arginine 31 facilitates greater conformational change during binding. This work describes the role of a somatic mutation to arginine in the binding pocket, and suggests how a structural scaffold such as an antibody can evolve to achieve sequence-specificity and pathogenicity in lupus. |
