The 'wildtype' conformation of p53: epitope mapping using hybrid proteins.
The function of p53 correlates with its 'wildtype' conformation, specifically recognized by antibodies PAb1620 and PAb246, and many cancer-associated mutations cause loss of this conformation. The epitopes of these antibodies were identified using hybrid p53 proteins created by a new method. Plasmids carrying homologous genes cut at appropriate sites recombined efficiently when transformed into RecE(+) E. coli. PAb1620 and PAb246 recognize mouse but not chicken p53; we created mouse-chicken hybrids of the p53 core domain and tested antibody reactivity. PAb246 binding mapped to residues 201-212, while PAb1620 required both residues 145-157 and 201-212 (human p53 numbering used throughout). An alanine-scan showed that the key residues for PAb246 and PAb1620 are completely distinct: PAb246 recognizes residues 202-204 (Tyr-Pro-Glu) while PAb1620 recognizes residues Arg156, Leu206, Arg209, and Gln/Asn210, the last two residues being essential. Both antibody epitopes are far from the p53 interface with DNA, but near the epitope of the 'mutant' conformation antibody PAb240. These epitope locations may help in dissecting the interactions of p53, including those with E6/E6-AP and in its DNA-bound state.
Wang
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PL
Centre for Protein Engineering, Hills Road, Cambridge CB2 2QH, UK.
Sait
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Winter
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D002645:Chickens; D004247:DNA / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D016159:Tumor Suppressor Protein p53; D004247:DNA