Domain Organization of IκBα and Sites of Interaction with NF-κB p65

Abstract

The DNA-binding activity and cellular distribution of the transcription factor NF-κB are regulated by the inhibitor protein IκBα. IκBα belongs to a family of proteins that contain multiple repeats of a 30- to 35-amino-acid sequence that was initially recognized in the erythrocyte protein ankyrin. Partial proteolysis has been used to study the domain structure of IκBα and to determine the sites at which it interacts with NF-κB. The data reveal a tripartite structure for IκBα in which a central, protease-resistant domain composed of five ankyrin repeats is flanked by an unstructured N-terminal extension and a compact, highly acidic C-terminal domain that is connected to the core of the protein by a flexible linker. Functional analysis of V8 cleavage products indicates that IκBα molecules lacking the N-terminal region can interact with and inhibit the DNA-binding activity of the p65 subunit of NF-κB, whereas IκBα molecules which lack both the N- and C-terminal regions are incapable of doing so. Protease cleavage of the N terminus of IκBα was unaffected by the presence of the p65 subunit of NF-κB, whereas bound p65 blocked cleavage of the flexible linker connecting the C-terminal domain to the ankyrin repeat-containing core of the protein. This linker region is highly conserved within the human, rat, pig, and chicken homologs of IκBα, and while it has been suggested that it represents a sixth ankyrin repeat, it is also likely that this is a flexible region of the protein that interacts with NF-κB.