Interactions between NFkappaB and its inhibitor ikappaB: biophysical characterization of a NFkappaB/ikappaB-alpha complex.
The N-terminal domain (1-318 amino acids) of mouse NFkappaB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length ikappaB-alpha (MAD3, 1-317 amino acids) molecule was generated by binding the E. coli-derived ikappaB-alpha to the purified NFkappaB and purifying the complex by sequential chromatography. The stoichiometry of NFkappaB to ikappaB in the complex was determined to be 2 to 1 by light scattering and SDS-polyacrylamide gel electrophoresis. The secondary structure of the NFkappaB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFkappaB occurs upon binding of DNA. The FTIR spectrum of the NFkappaB/ikappaB complex indicates that its secondary structure is composed of 17% alpha-helix, 39% beta-strand, 18% irregular structures, and 26% beta-turns and loops. By comparing these data to the FTIR data for NFkappaB alone, it is concluded that the ikappaB (MAD3) in the complex contains 35% alpha-helix, 27% beta-strand, 22% irregular structures, and 16% beta-turns and loops. Circular dichroism (CD) analysis of a shorter form of ikappaB (pp40) indicates that it contains at least 20% alpha-helix and that the ikappaB subunit accounts for nearly all of the alpha-helix present in the NFkappaB/ikappaB complex, consistent with the FTIR results. The stabilities of NFkappaB, ikappaB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of ikappaB is enhanced upon the formation of the NFkappaB/ikappaB complex.
Li
T
T
Department of Small Molecule Chemistry and Process Science, Amgen Inc., Thousand Oaks, California 91320, USA.
Narhi
L O
LO
Wen
J
J
Philo
J S
JS
Sitney
K
K
Inoue
J
J
Yamamoto
T
T
Arakawa
T
T
D000818:Animals; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020672:I-kappa B Proteins; D008027:Light; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D012542:Scattering, Radiation; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature