High-affinity interaction between IKKβ and NEMO

The Ser/Thr-specific I kappa B kinase (IKK), which comprises IKK alpha or IKK beta and the regulatory protein NEMO, is at the bottleneck for NF-kappa B activation. IKK activity relies on interaction between NEMO and IKK alpha or IKK beta. A conserved region in the C-terminal tail of IKK beta or IKK alpha (NEMO-binding domain, NBD, residues 734-745 of IKK beta) is important for interaction with NEMO. Here we show that the NBD peptide of IKK beta is not sufficient for interaction with NEMO. Instead, a longer region of the IKK beta C-terminal region provides high affinity for NEMO. Quantitative measurements using surface plasmon resonance and isothermal titration calorimetry confirm the differential affinities of these interactions and provide insight into the kinetic and thermodynamic behaviors of the interactions. Biochemical characterization using multiangle light scattering (MALS) coupled with refractive index shows that the longer IKK beta C-terminal region forms a 2:2 stoichiometirc complex with NEMO.