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BACKGROUND: The MDM2 oncoprotein binds to the tumor suppressor p53 and inhibits its anti-oncogenic functions. MATERIALS AND METHODS: To determine the amino acids of MDM2 that are critical for binding to p53, a modified two-hybrid screen was performed in yeast. Site-directed mutagenesis was then performed to identify MDM2 residues important for p53 interaction. Mutant MDM2 proteins were subsequently tested for their ability to bind to p53 in vitro and for their ability to regulate p53-mediated transcription in vivo. RESULTS: The yeast genetic screen yielded two Mdm2 mutations (G58D and C77Y) which disrupted binding to p53 in vitro without altering the conformation of MDM2 as determined with conformation-sensitive monoclonal antibodies. Site-directed mutagenesis yielded mutations of two additional amino acids 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 consistent with prior indications that a physical interaction between the two proteins is required for MDM2's inhibition of p53. Finally, the crystal structure of the MDM2-p53 complex shows that two of the four critical residues identified here contact p53 directly, while the remaining two residues play important structural roles in the MDM2 domain. CONCLUSIONS: MDM2 residues G58, D68, V75, and C77 are critical for MDM2's interaction with the p53 protein. Mutation of these residues to alanine prevents MDM2's interaction with p53 in vitro, and MDM2's regulation of p53's transcriptional activity in vivo.