STD and TRNOESY NMR studies for the epitope mapping of the phosphorylation motif of the oncogenic protein β-catenin recognized by a selective monoclonal antibody

AbstractThe interaction of the P-β-Cat19–44 peptide, a 26 amino acid peptide (K19AAVSHWQQQSYLDpSGIHpSGATTTAP44) that mimics the phosphorylated β-Catenin antigen, has been studied with its monoclonal antibody BC-22, by transferred nuclear Overhauser effect NMR spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) spectroscopy. This antibody is specific to diphosphorylated β-Catenin and does not react with the non-phosphorylated protein. Phosphorylation of β-Catenin at sites Ser33 and Ser37 on the DSGXXS motif is required for the interaction of β-Catenin with the ubiquitin ligase SCFβ-TrCP. β-TrCP is involved in the ubiquitination and proteasome targeting of the oncogenic protein β-Catenin, the accumulation of which has been implicated in various human cancers. The three-dimensional structure of the P-β-Cat19–44 in the bound conformation was determined by TRNOESY NMR experiments; the peptide adopts a compact structure in the presence of mAb with formation of turns around Trp25 and Gln26, with a tight bend created by the DpS33GIHpS37 motif; the peptide residues (D32-pS37) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide’s epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentate association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of β-Catenin with the SCFβ-TrCP protein