| Abstract
| - ATF4 plays a crucial role in the cellular response to stress. The E3 ubiquitin ligase, SCFβ-TrCP protein responsible for ATF4 degradation by the proteasome, binds to ATF4 through a DpSGXXXpSphosphorylation motif, which is similar but not identical to the DpSGXXpS motif found in most othersubstrates of β-TrCP. NMR studies were performed on the free and bound forms of a peptide derivedfrom this ATF4 motif that enabled the elucidation of the conformation of the ligand complexed to theβ-TrCP protein and its binding mode. Saturation transfer difference (STD) NMR allowed the study ofcompetition for binding to β-TrCP, between the phosphorylation motifs of ATF4 and β-catenin, tocharacterize the ATF4 binding epitope. Docking protocols were performed using the crystal structure ofthe β-catenin−β-TrCP complex as a template and NMR results of the ATF4−β-TrCP complex. Inagreement with the STD results, in order to bind to β-TrCP, the ATF4 DpSGIXXpSXE motif requiredthe association of two negatively charged areas, in addition to the hydrophobic interaction in the β-TrCPcentral channel. Docking studies showed that the ATF4 DpSGIXXpSXE motif fits the binding pocket ofβ-TrCP through an S-turning conformation. The distance between the two phosphate groups is 17.8 Å,which matched the corresponding distance 17.1 Å for the other extended DpSGXXpS motif in the β-TrCPreceptor model. This study identifies the residues of the β-TrCP receptor involved in ligand recognition.Using a new concept of STD competition experiment, we show that ATF4 competes and inhibits bindingof β-catenin to β-TrCP.
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