A GENETIC APPROACH TO MAPPING THE P53 BINDING-SITE IN THE MDM2 PROTEIN

Background: The MDM2 oncoprotein binds to the tumor suppressor p53 and inhibits its anti-ontogenic functions. Materials and Methods: To dete rmine the amino acids of MDM2 that are critical for binding to p53, a modified two-hybrid screen was performed in yeast. site-directed mutag enesis was then performed to identify MDM2 residues important for p53 interaction. Mutant MDMZ proteins were subsequently tested for their a bility to bind to p53 in vitro and for their ability to regulate p53-m ediated transcription in vivo. Results: The yeast genetic screen yield ed two Mdm2 mutations (G58D and C77Y) which disrupted binding to p53 i n vitro without altering the conformation of MDM2 as determined with c onformation-sensitive monoclonal antibodies. Site-directed mutagenesis yielded mutations of two additional amino adds of MDM2 (D68 and V75) that prevented binding to p53 in vitro. The mutant MDM2 proteins were unable to inhibit p53-dependent transcription in vivo, which is consis tent with prior indications that a physical interaction between the tw o proteins is required for MDM2's inhibition of p53. Finally, the crys tal structure of the MDM2-p53 complex shows that two of the four criti cal residues identified here contact p53 directly, while the remaining two residues play important structural roles in the MDM2 domain. Conc lusions: MDM2 residues G58, D68, V75, and C77 are critical for MDM2's interaction with the p53 protein. Mutation of these residues to alanin e prevents MDM2's interaction with p53 in vitro, and MDM2's regulation of p53's transcriptional activity in vivo.