Transfer-NMR and Docking Studies Identify the Binding of the Peptide Derived from Activating Transcription Factor 4 to Protein Ubiquitin Ligase β-TrCP. Competition STD-NMR with β-Catenin

Abstract

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.

Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/β-TrCP Axis

“…The method of purification of MBP-␤-TrCP was previously described (36). Following this protocol, the final yield of purified MBP-␤-TrCP was 1.3 mg/liter.…”

“…The calculated distances were incorporated into a simulated annealing protocol within the program ARIA 2.3 (25). Details on the three-dimensional structure calculation were presented elsewhere (36). PyMOL (http://www.pymol.org) was used for the analysis and presentation of the results of structure determination.…”

βTrCP interacts with the ubiquitin-dependent endocytosis motif of the GH receptor in an unconventional manner

“…The crystal structure of a complex of βTrCP in complex with β-catenin was solved and revealed the characteristics of this interaction in molecular detail [20]. Additionally, NMR studies have characterized the interaction of βTrCP with classical phosphorylated degrons DSG(X) 2 + n S in several substrates [21][22][23][24][25][26]. For the binding of the GHR-DSGXXS to βTrCP, the same residues in the WD40 domain are involved.…”

STD NMR spectroscopy: a case study of fosfomycin binding interactions in living bacterial cells

“…The ligand's hydrogen most tightly bound to the macromolecule will receive the most intense magnetization-transfer and the amplitude of these signals will change accordingly to the nOe effects. 5,6 Therefore, the degree of nOe effects reflects the proximity of these protons to the macromolecule, allowing direct observation of the ligand moiety involved in the macromolecule-ligand interaction. Among the vast literature covering biological interactions observed by STD NMR spectroscopy there are few examples in which the detection of those binding processes occurs directly in whole living cells.…”