Structure, function, and aggregation of the zinc-free form of the p53 DNA binding domain.
The p53 DNA binding domain (DBD) contains a single bound zinc ion that is essential for activity. Zinc remains bound to wild-type DBD at temperatures below 30 degrees C; however, it rapidly dissociates at physiological temperature. The resulting zinc-free protein (apoDBD) is folded and stable. NMR spectra reveal that the DNA binding surface is altered in the absence of Zn(2+). Fluorescence anisotropy studies show that Zn(2+) removal abolishes site-specific DNA binding activity, although full nonspecific DNA binding affinity is retained. Surprisingly, the majority of tumorigenic mutations that destabilize DBD do not appreciably destabilize apoDBD. The R175H mutation instead substantially accelerates the rate of Zn(2+) loss. A considerable fraction of cellular p53 may therefore exist in the folded zinc-free form, especially when tumorigenic mutations are present. ApoDBD appears to promote aggregation of zinc-bound DBD via a nucleation-growth process. These data provide an explanation for the dominant negative phenotype exhibited by many mutations. Through a combination of induced p53 aggregation and diminished site-specific DNA binding activity, Zn(2+) loss may represent a significant inactivation pathway for p53 in the cell.
Butler
James S
JS
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, New York 13210, USA.
Loh
Stewart N
SN
D001059:Apoproteins / Q000737:chemistry / Q000235:genetics; D001120:Arginine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D013329:Structure-Activity Relationship; D013696:Temperature; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics; D015032:Zinc / Q000737:chemistry*
D001059:Apoproteins; D004268:DNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D001120:Arginine; D015032:Zinc; D005998:Glycine