High-Affinity Interaction between IKKβ and NEMO

Abstract

The Ser/Thr-specific IκB kinase (IKK), which comprises IKKα or IKKβ and the regulatory protein NEMO, is at the bottleneck for NF-κB activation. IKK activity relies on interaction between NEMO and IKKα or IKKβ. A conserved region in the C-terminal tail of IKKβ or IKKα (NEMO-binding domain, NBD, residues 734–745 of IKKβ) is important for interaction with NEMO. Here we show that the NBD peptide of IKKβ is not sufficient for interaction with NEMO. Instead, a longer region of the IKKβ 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 IKKβ C-terminal region forms a 2:2 stoichiometirc complex with NEMO.