STD and TRNOESY NMR studies for the epitope mapping of the phosphorylation motif of the oncogenic protein beta-catenin recognized by a selective monoclonal antibody.
The interaction of the P-beta-Cat(19-44) peptide, a 26 amino acid peptide (K(19)AAVSHWQQQSYLDpSGIHpSGATTTAP(44)) that mimics the phosphorylated beta-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 beta-Catenin and does not react with the non-phosphorylated protein. Phosphorylation of beta-Catenin at sites Ser33 and Ser37 on the DSGXXS motif is required for the interaction of beta-Catenin with the ubiquitin ligase SCF(beta-TrCP). beta-TrCP is involved in the ubiquitination and proteasome targeting of the oncogenic protein beta-Catenin, the accumulation of which has been implicated in various human cancers. The three-dimensional structure of the P-beta-Cat(19-44) 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 DpS(33)GIHpS(37) 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 beta-Catenin with the SCF(beta-TrCP) protein.
Megy
Simon
S
Université Paris V-René Descartes, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, 45 Rue des Saint-Pères, 75270 Paris Cedex 06, France.
Bertho
Gildas
G
Gharbi-Benarous
Josyane
J
Baleux
Françoise
F
Benarous
Richard
R
Girault
Jean-Pierre
JP
D020816:Amino Acid Motifs; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism; D009369:Neoplasms / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D044843:SKP Cullin F-Box Protein Ligases / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D051176:beta Catenin / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009363:Neoplasm Proteins; D010455:Peptides; D025801:Ubiquitin; D051176:beta Catenin; D044843:SKP Cullin F-Box Protein Ligases