CONFORMATIONAL-CHANGES IN P53 ANALYZED USING NEW ANTIBODIES TO THE CORE DNA-BINDING DOMAIN OF THE PROTEIN

The p53 protein contains a protease resistant core section that binds to DNA in a sequence specific manner and whose crystal structure has b een determined. This core is flanked at the N-terminus by the transcri ptional transactivation domain and at the C-terminus by sequences invo lved in the oligomerisation of the protein. Extensive immunochemical a nalysis of p53 has shown that dominant antigenic sites lie within thes e N- and C-terminal domains while few antibodies to the central core h ave been identified; One of these, PAb240, has been extensively charac terised as its epitope is cryptic in the native DNA binding core struc ture but is exposed by denaturation. This epitope is also exposed on m any p53 proteins that contain point mutations in the core domain sugge sting that these mutations may have a common affect on the structure o f the core. To investigate this further we have generated several new antibodies to novel sites on p53 and mapped their epitopes using synth etic peptides. We find that antibodies to two other discrete sites in the core can also, like PAb240, recognize cryptic epitopes and disting uish mutant from wild-type conformations implying that the point mutat ions found in p53 in human tumours have widespread effects on the fold ing pattern of the DNA binding domain.