Transfer-NMR and docking studies identify the binding of the peptide derived from activating transcription factor 4 to protein ubiquitin ligase beta-TrCP. Competition STD-NMR with beta-catenin.
ATF4 plays a crucial role in the cellular response to stress. The E3 ubiquitin ligase, SCF beta-TrCP protein responsible for ATF4 degradation by the proteasome, binds to ATF4 through a DpSGXXXpS phosphorylation motif, which is similar but not identical to the DpSGXXpS motif found in most other substrates of beta-TrCP. NMR studies were performed on the free and bound forms of a peptide derived from this ATF4 motif that enabled the elucidation of the conformation of the ligand complexed to the beta-TrCP protein and its binding mode. Saturation transfer difference (STD) NMR allowed the study of competition for binding to beta-TrCP, between the phosphorylation motifs of ATF4 and beta-catenin, to characterize the ATF4 binding epitope. Docking protocols were performed using the crystal structure of the beta-catenin-beta-TrCP complex as a template and NMR results of the ATF4-beta-TrCP complex. In agreement with the STD results, in order to bind to beta-TrCP, the ATF4 DpSGIXXpSXE motif required the association of two negatively charged areas, in addition to the hydrophobic interaction in the beta-TrCP central channel. Docking studies showed that the ATF4 DpSGIXXpSXE motif fits the binding pocket of beta-TrCP through an S-turning conformation. The distance between the two phosphate groups is 17.8 A, which matched the corresponding distance 17.1 A for the other extended DpSGXXpS motif in the beta-TrCP receptor model. This study identifies the residues of the beta-TrCP receptor involved in ligand recognition. Using a new concept of STD competition experiment, we show that ATF4 competes and inhibits binding of beta-catenin to beta-TrCP.
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Université Paris Descartes, UMR 8601 CNRS, 45 rue des Saint-Pères, 75270 Paris Cedex 06, France.
Julien
J
Pons
Nathalie
N
Evrard-Todeschi
Gildas
G
Bertho
Josyane
J
Gharbi-Benarous
Valérie
V
Tanchou
Richard
R
Benarous
Jean-Pierre
JP
Girault
D051701:Activating Transcription Factor 4 / Q000737:chemistry*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051176:beta Catenin / Q000737:chemistry* / Q000378:metabolism; D044844:beta-Transducin Repeat-Containing Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D010455:Peptides; D011993:Recombinant Fusion Proteins; D051176:beta Catenin; D044844:beta-Transducin Repeat-Containing Proteins; D051701:Activating Transcription Factor 4