The IkappaBalpha/NF-kappaB complex has two hot spots, one at either end of the interface.
IkappaBalpha binds to and inhibits the transcriptional activity of NF-kappaB family members via its ankyrin repeat (AR) domain. The binding affinity of IkappaBalpha with NF-kappaB(p50/p65) heterodimers and NF-kappaB(p65/65) homodimers is in the picomolar range, and in the cell, this results in long half-lives of the complexes. Direct binding experiments have been performed using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) on a series of truncations and mutations in order to understand what regions of the interface are most important for the tight binding affinity of this complex. We previously showed that interactions between residues 305 and 321 of NF-kappaB(p65) with the first AR of IkappaBalpha are critical for the binding energy. Interactions in this region are responsible for more than 7 kcal/mol of the binding energy. Here we show equally drastic consequences for the binding energy occur upon truncation of even a few residues at the C terminus of IkappaBalpha. Thus, the interface actually has two hot spots, one at either end of the elongated and large surface of interaction. These results suggest a "squeeze" mechanism that leads to the extremely high affinity of the IkappaBalpha*NF-kappaB complex through stabilization of the ankyrin repeat domain.
Bergqvist
Simon
S
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0378, USA.
Ghosh
Gourisankar
G
Komives
Elizabeth A
EA
D000595:Amino Acid Sequence; D017089:Ankyrin Repeat / Q000235:genetics / Q000502:physiology*; D002151:Calorimetry / Q000379:methods; D019281:Dimerization; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D020349:Surface Plasmon Resonance / Q000379:methods; D013816:Thermodynamics
D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha