Direct observation of a transient ternary complex during IκBα-mediated dissociation of NF-κB from DNA

Abstract

We previously demonstrated that IκBα markedly increases the dissociation rate of DNA from NF-κB. The mechanism of this process remained a puzzle because no ternary complex was observed, and structures show that the DNA and IκBα binding sites on NF-κB are overlapping. The kinetics of interaction of IκBα with NF-κB and its complex with DNA were analyzed by using stopped-flow experiments in which fluorescence changes in pyrene-labeled DNA or the native tryptophan in IκBα were monitored. Rate constants governing the individual steps in the reaction were obtained from analysis of the measured rate vs. concentration profiles. The NF-κB association with DNA is extremely rapid with a rate constant of 1.5 × 10 8 M , yielding a K D of 2.8 nM. When IκBα is added to the NF-κB-DNA complex, we observe the formation of a transient ternary complex in the first few milliseconds of the fluorescence trace, which rapidly rearranges to release DNA. The rate constant of this IκBα-mediated dissociation is nearly equal to the rate constant of association of IκBα with the NF-κB-DNA complex, showing that IκBα is optimized to repress transcription. The rate constants for the individual steps of a more folded mutant IκBα were also measured. This mutant associates with NF-κB more rapidly than wild-type IκBα, but it associates with the NF-κB-DNA complex more slowly and also is less efficient at mediating dissociation of the NF-κB-DNA complex.

Molecular stripping in the NFκB/IκB/DNA genetic regulatory network

“…3B), shows that there is a larger barrier for releasing IκB from the complex than for releasing DN A. This is in harmony with kinetic mixing experiments that do not observe dissociation of the IκB on the laboratory time scale (7,12). Combining the two 1D Free energy profiles into a two dimensional free energy surface (Fig.…”

“…Recent in vitro kinetic measurements of IκB binding to the complex of N F κB with DNA challenge this equilibrium view. N F κB dissociates from DNA in an IκB concentration dependent manner (7,17). Including this enhancement 3 peer-reviewed) is the author/funder.…”

“…This classic picture, with the law of mass action at its core (5,6), suggests that understanding the molecular mechanism of the binding and release of transcription factors is of secondary interest compared with understanding the thermodynamics of protein-DNA recognition. The recent discovery of protein induced release of a DN A-bound transcription factor in the N F κB/IκB/DN A genetic switch changes this picture (7). The induced process, called "molecular stripping", opens up the possibility of molecular kinetic control of binding and release thus overturning the classical paradigm based only on thermodynamic control.…”

Functional importance of stripping in NFκB signaling revealed by a stripping-impaired IκBα mutant

“…A very transient NFκB-DNA-IκBα complex was indeed observed in stopped-flow fluorescence experiments (11). At high concentrations, signals corresponding to a ternary NFκB-DNA-IκBα complex were also observed by NMR (12,13).…”

“…The average rate of GFP-NFκB nuclear export for IκBα(WT) was 2.0 (± 0.2) × 10 −2 /min, whereas the IκBα(5xPEST) transfectants had a ninefold slower rate of GFP-NFκB nuclear export of 2.2 (± 0.1) × 10 −3 /min. Discussion IκBα(WT) efficiently strips NFκB from DNA (6,11). The mechanism of this process necessitates the formation of a ternary complex, which was extremely transient under stopped-flow conditions but could be observed under the high-concentration conditions of NMR experiments (11)(12)(13).…”