Characterization of baculovirus recombinant wild-type p53. Dimerization of p53 is required for high-affinity DNA binding and cysteine oxidation inhibits p53 DNA binding.
A high-yield, rapid and non-denaturing purification protocol for baculovirus recombinant wild-type p53 is described. Gel-filtration chromatography and chemical cross-linking experiments indicated that purified p53 assembles into multimeric forms ranging from tetramer to higher oligomers. A gel-mobility-shift assay and protein-DNA cross-linking studies demonstrated that purified baculovirus recombinant p53 binds to consensus DNA target as a dimer but that additional p53 molecules may then associate with the preformed p53-dimer-DNA complexes to form larger p53 DNA complexes. These observations suggest that the p53 tetramers and higher oligomers that form the minimal p53 association in solution dissociate upon DNA binding to form p53 dimer-DNA complexes. Binding of the mAB PAb 421 to the oligomerization-promoting domain on p53 stimulated sequentially formation of both p53-dimer-DNA and larger p53-DNA complexes. This observation suggests that factors may exist in vivo that could participate in the formation and the stabilization of the various p53-DNA complexes. Further characterization of the purified p53 revealed that the protein possesses highly reactive cysteine residues. We show that intrachain disulfide bonds form within the purified p53 molecules during storage in the absence of reducing agent. Zn2+ binding to p53 protect sulfhydryl groups from oxidation. Cysteine oxidation by intramolecular disulfide-bond formation did not modify the wild-type immunoreactive phenotype of the p53 protein but totally inhibited its DNA-binding activities. The oxidation of the p53 cysteine residues was also observed for nuclear p53 in baculovirus-infected insect cells. The redox status of the nuclear p53 regulates its DNA-binding activity in vitro confirming the essential role of the reduced state of cysteine residues in p53 for detectable DNA-binding activity.
Delphin
C
C
Laboratoire de Biologie Moleculaire du Cycle Cellulaire, INSERM Unite 309, Commissariat à l'Energie Atomique, Grenoble, France.
Cahen
P
P
Lawrence
J J
JJ
Baudier
J
J
D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D016159:Tumor Suppressor Protein p53; D004247:DNA; D003545:Cysteine