Characterization of the IKK Signaling Complex

Abstract

The transcription factor NF-κB is critical to the coordination of both innate and adaptive immune responses. The IκB kinase (IKK) complex is an important regulator of NF-κB activity. Targets that affect NF-κB signaling represent attractive therapeutic strategies for autoimmune diseases. Understanding assembly of the IKK complex is a necessary step in elucidating the molecular function of this complex and provides insight on methods of potential therapeutic intervention. The IKK complex is composed of three subunits: IKKα, IKKβ and NEMO (IKKγ). In this paper analytical ultracentrifugation is used to characterize the homo-oligomerization of the IKK components: α, β and γ as well as the hetero-oligiomerization of the components. The program MULTI-COIL was used to identify a C-terminal region of IKKβ which immediately precedes the NBD (NEMO binding domain) with a propensity to form a coiled-coil. The corresponding sequence from IKKα has a lower propensity. A fluorescent chimeric peptide was made which contains the predicted IKKβ coiled-coil and the IKKα NBD. This peptide binds to a construct containing the first 200 residues of NEMO with a Kd of 90 nM. Fluorescence polarization studies show that IKKβ binds to NEMO with a similar affinity. The affinities of IKKα as well as a synthetic NBD peptide are much lower. We conclude that a coiled-coil interaction in conjunction with the NBD contribute to IKK-NEMO binding.