High-affinity interaction between IKKbeta and NEMO.
The Ser/Thr-specific IkappaB kinase (IKK), which comprises IKKalpha or IKKbeta and the regulatory protein NEMO, is at the bottleneck for NF-kappaB activation. IKK activity relies on interaction between NEMO and IKKalpha or IKKbeta. A conserved region in the C-terminal tail of IKKbeta or IKKalpha (NEMO-binding domain, NBD, residues 734-745 of IKKbeta) is important for interaction with NEMO. Here we show that the NBD peptide of IKKbeta is not sufficient for interaction with NEMO. Instead, a longer region of the IKKbeta 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 IKKbeta C-terminal region forms a 2:2 stoichiometirc complex with NEMO.
Lo
Yu-Chih
YC
Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.
Maddineni
Upendra
U
Chung
Jee Y
JY
Rich
Rebecca L
RL
Myszka
David G
DG
Wu
Hao
H
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051550:I-kappa B Kinase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
C491731:IKBKG protein, human; D051550:I-kappa B Kinase