Mdm2 binding to a conformationally sensitive domain on p53 can be modulated by RNA

Biochemical characterisation of the interaction of mdm2 protein with p53 pr otein has demonstrated that full-length mdm2 does not bind stably to p53-DN A complexes, contrasting with C-terminal truncations of mdm2 which do bind stably to p53-DNA complexes, In addition, tetrameric forms of the p53His175 mutant protein in the PAb1620+ conformation are reduced in binding to mdm2 protein. These data suggest that the mdm2 binding site in the BOX-I domain of p53 becomes concealed when either p53 binds to DIVA or when the core do main of p53 is unfolded by missense mutation. This further suggests that th e C-terminus of mdm2 protein contains a negative regulatory domain that aff ects mdm2 protein binding to a second, conformationally sensitive interacti on site in the core domain of p53. We investigated whether there was a seco nd docking site on p53 for mdm2 protein by examining the interaction of ful l-length mdm2 with p53 lacking the BOX-I domain. Although mdm2 protein did bind very weakly to p53 protein lacking the BOX-I domain, addition of RNA a ctivated mdm2 protein binding to this truncated form of p53, These data pro vide evidence for three previously undefined regulatory stages in the p53-m dm2 binding reaction: (1) conformational changes in p53 protein due to DNA binding or point mutation conceals a secondary docking site of mdm2 protein ; (2) the C-terminus of mdm2 is the primary determinant which confers this property upon mdm2 protein; and (3) mdm2 protein binding to this secondary interaction site within p53 can be stabilised by RNA. (C) 2000 Federation o f European Biochemical Societies.