Thermodynamics reveal that helix four in the NLS of NF-kappaB p65 anchors IkappaBalpha, forming a very stable complex.
IkappaBalpha is an ankyrin repeat protein that inhibits NF-kappaB transcriptional activity by sequestering NF-kappaB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IkappaBalpha ankyrin repeat domain to NF-kappaB(p50/p65) using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR data showed that the IkappaBalpha and NF-kappaB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a K(D,obs) of approximately 40 pM at 37 degrees C. As reported previously, the amino-terminal DNA-binding domain of p65 contributes little to the overall binding affinity. Conversely, helix four of p65, which forms part of the nuclear localization sequence, was essential for high-affinity binding. This was surprising, given the small size of the binding interface formed by this part of p65. The NF-kappaB(p50/p65) heterodimer and p65 homodimer bound IkappaBalpha with almost indistinguishable thermodynamics, except that the NF-kappaB p65 homodimer was characterized by a more favorable DeltaH(obs) relative to the NF-kappaB(p50/p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (DeltaC(P,obs)), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be accounted for readily by the small loss of buried non-polar surface area and we hypothesize that the observed effect is due to additional folding of some regions of the complex.
Bergqvist
Simon
S
Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0378, USA.
Croy
Carrie H
CH
Kjaergaard
Magnus
M
Huxford
Tom
T
Ghosh
Gourisankar
G
Komives
Elizabeth A
EA
D000818:Animals; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D052002:NF-kappa B p50 Subunit / Q000737:chemistry / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D020349:Surface Plasmon Resonance; D013696:Temperature; D013816:Thermodynamics; D051996:Transcription Factor RelA / Q000737:chemistry* / Q000378:metabolism*
D020672:I-kappa B Proteins; D046912:Multiprotein Complexes; D052002:NF-kappa B p50 Subunit; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D019913:Nuclear Localization Signals; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA