Structural studies on 24P-IkappaBalpha peptide derived from a human IkappaB-alpha protein related to the inhibition of the activity of the transcription factor NF-kappaB.

The I kappa B-alpha protein, inhibitor of the transcription factor nuclear factor-kappa B (NF-kappa B), is a cellular substrate of beta-transducin repeat containing protein (beta-TrCP). beta-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. beta-TrCP targets the protein I kappa B-alpha for ubiquitination, followed by proteasome degradation. The SCF-beta-TrCP complex specifically recognizes an I kappa B-alpha peptide containing the DpSGXXpS motif in a phosphorylation-dependent manner. A fragment comprising 24 amino acids residues for the phosphorylated peptide at the two sites Ser32 and Ser36 and thus termed 24P-I kappa B alpha (P-I kappa B alpha(21-44)) was characterized conformationally by NMR spectroscopy and molecular dynamics simulation. In the free states, 24P-I kappa B alpha exhibits mainly a random coil conformation, although the presence of a nascent bend was detected between residues 30 and 36, flanked by two N- and C-terminal disordered regions. The bound conformation of the phosphorylated I kappa B-alpha peptide was obtained using transfer nuclear Overhauser effect spectroscopy (TRNOESY) experiments. To further elucidate the basis of the beta-TrCP interaction, a complex between 24P-I kappa B alpha peptide and beta-TrCP protein was studied using saturation transfer difference (STD) NMR experiments. The conformation of 24P-I kappa B alpha bound to beta-TrCP presents a bend corresponding to the (31)DpSGLDpS(36) motif and on both sides N- and C-terminal turn regions (Lys22-Asp31 and Met37-Glu43). The bound structure of the phosphorylated peptide suggests that these domains are crucial for the interaction of the peptide with its receptor showing the protons identified by STD NMR as exposed in close proximity to the beta-TrCP surface.