Interactions between NFkappaB and its inhibitor ikappaB: biophysical characterization of a NFkappaB/ikappaB-alpha
1Department of Small Molecule Chemistry and Process Science, Amgen Inc., Thousand Oaks, California 91320, USA.
Insights
Researchers purified the N-terminal domain of mouse NFkappaB (p65) and studied its complex with ikappaB-alpha. The complex showed enhanced thermal stability of ikappaB upon formation, revealing key structural insights.
Area of Science:
- Molecular Biology
- Protein Biochemistry
- Structural Biology
Background:
- NFkappaB (p65) is a key transcription factor involved in immune responses.
- NFkappaB signaling is regulated by inhibitors of NFkappaB (IkappaB) proteins.
- Understanding the structural basis of NFkappaB/IkappaB interaction is crucial for drug development.
Purpose of the Study:
- To purify and characterize the N-terminal domain of mouse NFkappaB (p65).
- To generate and analyze the complex of NFkappaB (p65) with full-length IkappaB-alpha (MAD3).
- To investigate the structural and thermal properties of the NFkappaB/IkappaB complex.
Main Methods:
- Protein purification from Escherichia coli.
- Complex formation and purification using sequential chromatography.
- Stoichiometry determination by light scattering and SDS-PAGE.
- Secondary structure analysis using Fourier-transform infrared (FTIR) spectroscopy.
- Thermal stability assessment using Circular Dichroism (CD) spectroscopy.
Main Results:
- Homogeneous N-terminal domain of mouse NFkappaB (p65) was purified.
- A 2:1 stoichiometry of NFkappaB to IkappaB was determined in the complex.
- FTIR analysis revealed the secondary structure composition of NFkappaB, IkappaB, and their complex.
- CD analysis indicated that IkappaB contributes significantly to the alpha-helix content of the complex.
- The thermal stability of IkappaB was enhanced upon complex formation with NFkappaB.
Conclusions:
- The N-terminal domain of NFkappaB (p65) can be purified and forms a stable complex with IkappaB-alpha.
- The complex formation alters the secondary structure of IkappaB and enhances its thermal stability.
- These findings provide structural insights into NFkappaB regulation by IkappaB.
Abstract:
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.
