Sequence specific recognition of ssDNA by a lupus autoantibody: kinetics and mechanism of binding.
11F8 is a pathogenic anti-ssDNA monoclonal autoantibody isolated from a lupus-prone mouse. Previous studies have established that 11F8 is sequence specific. To determine the basis for the observed binding specificity, stopped-flow fluorescence spectroscopy was used to measure the kinetic parameters and establish the mechanisms for the association of 11F8 with its target sequence, noncognate, and nonspecific ssDNA ligands. The data revealed that sequence-specific binding follows a two-step mechanism where the initial association step is second order. Values of k(1) are fast and above the modified Smoluchowski limit for a diffusion limited interaction (10(5)-10(6)M(-1)s(-1)). The dependency of k(1) on [salt] and solvent polarity indicates that electrostatic steering is responsible for this rapid association rate. The second association step is rate limiting and is characteristic of an isomerization process during which binding interfaces are optimized. This step apparently is driven by the desolvation of hydrophobic surfaces within the binding interface. The differences in the rate of dissociation for the various DNA ligands suggest that specificity is governed primarily through the dissociation of the final complexes.
Beckingham
J A
JA
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Glick
G D
GD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D000915:Antibody Affinity / Q000187:drug effects; D000937:Antigen-Antibody Reactions / Q000187:drug effects / Q000276:immunology; D001323:Autoantibodies / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001666:Binding Sites, Antibody; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000276:immunology*; D007700:Kinetics; D008177:Lupus Vulgaris / Q000276:immunology*; D051379:Mice; D012997:Solvents / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013696:Temperature
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D004277:DNA, Single-Stranded; D012997:Solvents