Inhibitor κB kinase β binding by inhibitor κB kinase γ

Activation of a large multisubunit protein kinase, called the inhibitor kappa B kinase (IKK) complex, is central to the induction of the family of transcription factors nuclear factor kappa B. IKK is comprised of two catalytic subunits, IKK alpha and IKK beta, and a regulatory IKK gamma subunit. It is known that the catalytic IKK beta and regulatory IKK gamma subunits associate through interactions mediated by the N-terminal region of IKK gamma and an 11-mer peptide located near the C-terminus of IKK beta. In this study, we have defined the minimal IKK gamma segment that binds IKK beta and determined the binding affinity of the IKK beta/IKK gamma complex. We identified that the N-terminal segment spanning residues 40-130 of IKK gamma binds the IKK beta C-terminal domain (residues 665-756) with K-d approximate to 25 nM. Several smaller N-terminal IKK gamma deletion mutants within the N-terminal 130 residues, although in some cases retained IKK beta binding activity, showed a tendency to aggregate and formed covalently linked complexes. However, expansion of the C-terminus of these fragments to residue 210 completely changed the solution behavior of the IKK gamma N-terminus without affecting the IKK binding affinity. We also found that the IKK beta C-terminal domain formed a dimer in solution and the basic unit of the IKK beta/IKK gamma complex was a dimer/dimer.