Transdominant Inhibition by Tumor-derived p53 Mutants Tetramerization Domain of Wild-Type p53 and Alleviates An Engineered Four-Stranded Coiled Coil Substitutes for the

analysis of mutant GCN4 LZs3 has identified domains with altered subunit stoichiometries (41). One such engineered zipper, which will hereafter be referred to as the TZ, assembles as a parallel tetramer. This domain has not yet been shown to drive tetramerization of any protein. Furthermore, its ability to form tetramers in vivo has not been characterized. In an effort to generate a functional p53 protein that is not transdomi nantly inhibited by tumor-derived p53 mutants, we constructed and characterized a number of p53 proteins, the oligomerizalion of which is driven by the tetrameric or by the native GCN4 LZ. Our results indicate that the engineered TZ, but not the LZ, can drive p53 function in tumor cells and furthermore can alleviate transdominant inhibition by tumor derived p53 mutants. We have thus demonstrated the feasibility of designing a p53 protein with dominant wild-type function. MATERIALS AND METHODS Recombinant Plasmids. Standard cloning procedures were used, and all mutants were generated by PCR-directed mutagenesis (42). Plasmids pGEMhpS3wtB, pGEMhp53A344, and pGEMhp53LZ335Q encode human wild-type PS3, p53Ala344, and p53LZ335Q, respectively, and have been described (43). Plasmid pGEMhp53LZ343RMKQ encodes a hybrid protein consisting of residues 1†" 343 of human p53 and residues 249†" 281 of GCN4 (44). Because the LZ segment in p53LZ335Q refers to residues 253†" 281 of GCN4, for uniformity we consider p53LZ343RMKQ as having a LZ corre sponding to residues 253†" 281 of GCN4 and a linker consisting of residues 249†" 252 of GCN4, which are Arg-Met-Lys-Gln (RMKQ). Plasmid pGEMhp53TZ334NR was derived from pGEMhp53wtB by replacing the SstII-Sall fragment with synthetic oligonucleotides encoding the TZ (41) corresponding to residues 250†" 281 of GCN4 (44) via an Asn-Arg (NR) sequence. Plasmid pGEMhp53TZ334NRII352 was derived by inserting sequences encoding an Ile (I), followed by residues 352†" 393 of human p53 after the last codon in pGEMhp53TZ334NR. Plasmid pSV2 hp53wtB was prepared by cloning into the SaII-BglII sites of pSV2 humjun (45) a blunted EcoRI-HindIII p53 insert from pGEMbp53wtB (43). Plasmids expressing p53 fusion proteins were similarly derived from the corresponding pGEM plasmids. A pSV2 plasmid without insert was prepared by ligating pSV2 humjun (45) linearized with Sail and BglII. The tumor derived p53 mutation Trp248 was introduced in the context of pGEMhp53wtB and pSV2 hp53wtB by site-directed mutagenesis. Plasmids pEp2l/TKseap and pEmdm2lTKseap have one copy of oligonu cleotides Ep21 and Emdm2, respectively, cloned in the EcoRV site of …