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A Guide to Production, Crystallization, and Structure Determination of Human IKK1/α
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Mechanism of I kappa B alpha binding to NF-kappa B dimers

C B Phelps1, L L Sengchanthalangsy, T Huxford

  • 1Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA.

The Journal of Biological Chemistry
|July 7, 2000
PubMed

Insights

Nuclear factor kappa B (NF-kappa B) and I kappa B alpha binding is primarily driven by the NF-kappa B p65 subunit

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • NF-kappa B is a crucial transcription factor regulating immune responses and cell survival.
  • I kappa B alpha acts as a negative regulator of NF-kappa B, inhibiting its nuclear translocation.
  • Understanding their interaction is key to deciphering cellular signaling pathways.

Purpose of the Study:

  • To quantitatively assess the contributions of different protein-protein interactions to NF-kappa B.I kappa B alpha complex formation.
  • To identify specific regions and interactions responsible for binding affinity.
  • To investigate the role of post-translational modifications in regulating this complex.

Main Methods:

  • Gel electrophoretic mobility shift assay (EMSA) was used to measure binding affinities.
  • Analysis of X-ray crystal structures informed the investigation of interaction interfaces.
  • Oxidative cysteine cross-linking was employed to study dynamic interactions.

Main Results:

  • I kappa B alpha shows preferential binding to p50/p65 heterodimers and p65 homodimers over p50 homodimers.
  • The nuclear localization sequence (NLS) and C-terminal region of the NF-kappa B p65 subunit are major contributors to complex formation.
  • A secondary interaction between I kappa B alpha's acidic C-terminal/PEST-like region and the NF-kappa B p65 N-terminal domain also contributes to binding.
  • Phosphorylation of I kappa B alpha by casein kinase II enhances binding affinity.

Conclusions:

  • The NF-kappa B p65 subunit's NLS and adjacent regions are critical for high-affinity binding to I kappa B alpha.
  • Dynamic, non-specific interactions involving I kappa B alpha's acidic region also play a significant role.
  • Post-translational modification, specifically phosphorylation, modulates the binding strength of the NF-kappa B.I kappa B alpha complex.

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