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.
Mesh Terms:
Activating Transcription Factor 4, Amino Acid Motifs, Amino Acid Sequence, Humans, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Peptides, Phosphorylation, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Fusion Proteins, Sequence Homology, Amino Acid, beta Catenin, beta-Transducin Repeat-Containing Proteins
Activating Transcription Factor 4, Amino Acid Motifs, Amino Acid Sequence, Humans, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Peptides, Phosphorylation, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Fusion Proteins, Sequence Homology, Amino Acid, beta Catenin, beta-Transducin Repeat-Containing Proteins
Biochemistry
Date: Jan. 08, 2008
PubMed ID: 18052253
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