Structural and functional analysis of the NF-kappa B p65 C terminus. An acidic and modular transactivation domain with the potential to adopt an alpha-helical conformation.
The p65 subunit of the NF-kappa B transcription factor contains in its C-terminal 120 amino acids at least two transcription activation domains. One domain (TA1) is contained within only the 30 C-terminal amino acids. Structural studies employing CD and NMR spectroscopy revealed that the TA1 domain is unstructured. NMR analysis of a protein corresponding to the C-terminal 123 amino acids also showed a random coil conformation. However, a portion of TA1 was found to adopt an alpha-helical conformation in the presence of hydrophobic solvents. Transcriptional analysis of point mutants revealed the functional importance of two evolutionary conserved sequence repeats, which are located in the conditionally alpha-helical region of TA1. These repeats acted synergistically in transcription activation. The inhibitory effect of some mutants indicated secondary structure constraints on TA1 in intact cells. Inverting the sequence of two acidic activation domains significantly reduced their transactivating potential, suggesting that amino acid composition is not solely essential for activity; a defined primary structure is necessary as well. Acidic sequence motifs related in primary structure and squelching activity to those of TA1 are present in the activation domains of VP16, c-Rel, and several other transcription factors. We propose a model suggesting that primarily unstructured acidic activation domains can adopt a secondary structure upon contacting their target molecules by an "induced fit" mechanism.
Schmitz
M L
ML
Institute for Biochemistry, Albert Ludwigs University, Freiburg, Federal Republic of Germany.
dos Santos Silva
M A
MA
Altmann
H
H
Czisch
M
M
Holak
T A
TA
Baeuerle
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D005656:Fungal Proteins / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D020569:Proto-Oncogene Proteins c-rel; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000378:metabolism; D051996:Transcription Factor RelA; D015533:Transcriptional Activation
D005656:Fungal Proteins; C070633:Gal-VP16; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D011994:Recombinant Proteins; D015534:Trans-Activators; D051996:Transcription Factor RelA