Mdm2 binding to a conformationally sensitive domain on p53 can be modulated by RNA.
Biochemical characterisation of the interaction of mdm2 protein with p53 protein has demonstrated that full-length mdm2 does not bind stably to p53-DNA 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 DNA or when the core domain of p53 is unfolded by missense mutation. This further suggests that the C-terminus of mdm2 protein contains a negative regulatory domain that affects mdm2 protein binding to a second, conformationally sensitive interaction site in the core domain of p53. We investigated whether there was a second docking site on p53 for mdm2 protein by examining the interaction of full-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 activated mdm2 protein binding to this truncated form of p53. These data provide evidence for three previously undefined regulatory stages in the p53-mdm2 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.
Burch
L R
LR
Department of Molecular and Cellular Pathology, University of Dundee, Dundee, UK. lrburch@dundee.ac.uk
Midgley
C A
CA
Currie
R A
RA
Lane
D P
DP
Hupp
T R
TR
D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D020125:Mutation, Missense; D009687:Nuclear Proteins; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism
D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D012313:RNA; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2