Kinetic partitioning during folding of the p53 DNA binding domain.
The DNA-binding domain (DBD) of wild-type p53 loses DNA binding activity spontaneously at 37 degrees C in vitro, despite being thermodynamically stable at this temperature. We test the hypothesis that this property is due to kinetic misfolding of DBD. Interrupted folding experiments and chevron analysis show that native molecules are formed via four tracks (a-d) under strongly native conditions. Folding half-lives of tracks a-d are 7.8 seconds, 50 seconds, 5.3 minutes and more than five hours, respectively, in 0.3M urea (10 degrees C). Approximately equal fractions of molecules fold through each track in zero denaturant, but above 2.0M urea approximately 90% fold via track c. A kinetic mechanism consisting of two parallel folding channels (fast and slow) is proposed. Each channel populates an on-pathway intermediate that can misfold to form an aggregation-prone, dead-end species. Track a represents direct folding through the fast channel. Track b proceeds through the fast channel but via the off-pathway state. Track c corresponds to folding via the slow channel, primarily through the off-pathway state. Track d proceeds by way of an even slower, uncharacterized route. We postulate that activity loss is caused by partitioning to the slower tracks, and that structural unfolding limits this process. In support of this view, tumorigenic hot-spot mutants G245S, R249S and R282Q accelerate unfolding rates but have no affect on folding kinetics. We suggest that these and other destabilizing mutants facilitate loss of p53 function by causing DBD to cycle unusually rapidly between folded and unfolded states. A significant fraction of DBD molecules become effectively trapped in a non-functional state with each unfolding-folding cycle.
Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.
James S
JS
Butler
Stewart N
SN
Loh
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D020125:Mutation, Missense; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014508:Urea
D004268:DNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D014508:Urea