Kinetic analysis of sequence-specific recognition of ssDNA by an autoantibody.
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.
Beckingham
Jennifer A
JA
Department of Chemistry and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Cleary
Joanne
J
Bobeck
Melissa
M
Glick
Gary D
GD
D001323:Autoantibodies / Q000235:genetics / Q000276:immunology*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000276:immunology*; D007700:Kinetics; D016296:Mutagenesis; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013696:Temperature
D001323:Autoantibodies; D004277:DNA, Single-Stranded