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 IkappaB-alpha protein, inhibitor of the transcription factor nuclear factor-kappaB (NF-kappaB), 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 IkappaB-alpha for ubiquitination, followed by proteasome degradation. The SCF-beta-TrCP complex specifically recognizes an IkappaB-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-IkappaBalpha (P-IkappaBalpha21-44) was characterized conformationally by NMR spectroscopy and molecular dynamics simulation. In the free states, 24P-IkappaBalpha 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 IkappaB-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-IkappaBalpha peptide and beta-TrCP protein was studied using saturation transfer difference (STD) NMR experiments. The conformation of 24P-IkappaBalpha bound to beta-TrCP presents a bend corresponding to the 31DpSGLDpS36 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.
Pons
Julien
J
Université Paris V-René Descartes, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (UMR 8601 CNRS), 45 rue des Saint-Pères, 75270 Paris Cedex 06, France.
Evrard-Todeschi
Nathalie
N
Bertho
Gildas
G
Gharbi-Benarous
Josyane
J
Sonois
Vanessa
V
Benarous
Richard
R
Girault
Jean-Pierre
JP
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D018604:Epitope Mapping; D015497:HIV-1 / Q000378:metabolism; D054298:Human Immunodeficiency Virus Proteins; D006801:Humans; D020672:I-kappa B Proteins / Q000737:chemistry*; D008958:Models, Molecular; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism; D044844:beta-Transducin Repeat-Containing Proteins / Q000737:chemistry / Q000502:physiology; D000086082:Viroporin Proteins
D054298:Human Immunodeficiency Virus Proteins; D020672:I-kappa B Proteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D010446:Peptide Fragments; D054334:Viral Regulatory and Accessory Proteins; D044844:beta-Transducin Repeat-Containing Proteins; C000606204:NFKBIA protein, human; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins