Zn(2+)-dependent misfolding of the p53 DNA binding domain.
The DNA binding domain (DBD) of p53 folds by a complex mechanism that involves parallel pathways and multiple intermediates, both on- and off-pathway. This heterogeneity renders DBD particularly susceptible to misfolding and aggregation. The origins of parallel folding mechanisms are not well understood. DBD folding heterogeneity may be caused by the presence of the single bound Zn2+. To test that hypothesis, we carried out kinetic folding studies of DBD in its Zn2+-free form (apoDBD) and in the presence of various concentrations of free Zn2+ and the Zn2+-nitrilotriacetate (NTA) complex. Folding kinetics of apoDBD and DBD are similar, although apoDBD folds faster than DBD at some urea concentrations. The principle consequence of Zn2+ removal is to accelerate unfolding and simplify it from two exponential phases to one. Metal binding interactions are therefore not responsible for the observed complexity of the folding reaction. A slight stoichiometric excess of free Zn2+ arrests folding and traps the protein in a misfolded state in which Zn2+ is bound to nonphysiological ligands. Folding can be rescued by providing metal ions in the form of the NTA-Zn2+ complex, which simultaneously protects against misligation and provides a source of Zn2+ for regenerating the functional protein. This chemical metallochaperone strategy may be an effective means for improving folding efficiency of other metal binding proteins. The findings suggest that, in vivo, DBD must fold in an environment where free Zn2+ concentration is low and its bioavailability is carefully regulated by cellular metallochaperones.
Butler
James S
JS
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, New York 13210, USA.
Loh
Stewart N
SN
D001682:Biological Availability; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*; D015032:Zinc / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA; D015032:Zinc