Conformational changes induced by DNA binding of NF-kappa B

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Nuclear Factor kappa B (NF-kappa B) is a crucial transcription factor involved in immune responses and gene regulation.
• NF-kappa B forms extensive contacts with its DNA recognition site, involving structural transitions in both protein and DNA.
• Understanding these structural dynamics is key to elucidating NF-kappa B's regulatory mechanisms.

Purpose of the Study:

• To investigate the structural transitions in the NF-kappa B p50 subunit and its DNA upon complex formation.
• To determine how DNA binding by NF-kappa B affects the protein's conformation and protease sensitivity.
• To analyze DNA conformational changes induced by NF-kappa B binding.

Main Methods:

• Proteinase sensitivity assays (using chymotrypsin, proteinase K, and trypsin) to map cleavage sites on the p50 subunit.
• Circular dichroism (CD) spectroscopy to analyze conformational changes in both the p50 protein and DNA.
• Examination of near-UV CD spectra to compare bound and free DNA conformations.

Main Results:

• DNA binding blocked specific proteinase K, chymotrypsin, and trypsin cleavage sites (Y60, S74, K77, N78) in the p50 N-terminal AB loop.
• DNA binding enhanced cleavage at site Q180, located in a different region of the p50 subunit.
• CD spectroscopy revealed conformational changes in p50/p65 homodimers upon DNA binding.
• Specific DNA sequences, particularly high-affinity sites, exhibited altered conformations when bound to NF-kappa B.

Conclusions:

• The p50 N-terminal AB loop is unstructured in the absence of DNA but becomes rigid and proteinase-resistant upon DNA binding.
• NF-kappa B binding induces significant conformational changes in both the protein and, depending on the DNA sequence, the DNA itself.
• These findings highlight the dynamic nature of transcription factor-DNA interactions and their impact on protein structure and function.