ATP Binding to Hsp90 Is Sufficient for Effective Chaperoning of p53 Protein

Dawid Walerych,Malgorzata Gutkowska,Marcin P Klejman,Bartosz Wawrzynow,Zuzanna Tracz,Milena Wiech,Maciej Zylicz,Alicja Zylicz

doi:10.1074/jbc.m110.112110

Abstract

Hsp90 is a ubiquitous, ATP-dependent chaperone, essential for eukaryotes. It possesses a broad spectrum of substrates, among which is the p53 transcription factor, encoded by a tumor-suppressor gene. Here, we elucidate the role of the adenine nucleotide in the Hsp90 chaperone cycle, by taking advantage of a unique in vitro assay measuring Hsp90-dependent p53 binding to the promoter sequence. E42A and D88N Hsp90β variants bind but do not hydrolyze ATP, whereas E42A has increased and D88N decreased ATP affinity, compared with WT Hsp90β. Nevertheless, both of these mutants interact with WT p53 with a similar affinity. Surprisingly, in the case of WT, but also E42A Hsp90β, the presence of ATP stimulates dissociation of Hsp90-p53 complexes and results in p53 binding to the promoter sequence. D88N Hsp90β is not efficient in both of these reactions. Using a trap version of the chaperonin GroEL, which irreversibly captures unfolded proteins, we show that Hsp90 chaperone action on WT p53 results in a partial unfolding of the substrate. The ATP-dependent dissociation of p53-Hsp90 complex allows further folding of p53 protein to an active conformation, able to bind to the promoter sequence. Furthermore, in support of these results, the overproduction of WT or E42A Hsp90β stimulates transcription from the WAF1 gene promoter in H1299 cells. Altogether, our research indicates that ATP binding to Hsp90β is a sufficient step for effective WT p53 client protein chaperoning.

Highlights

Science and Higher Education. □S The on-line version of this article contains supplemental Experimental Procedures, references, and Figs. 1 and 2. 1 To whom correspondence may be addressed: International Institute of Molecular and Cell Biology in Warsaw, Ks
Despite the initial controversy on the ATP dependence of Hsp90 [12], it was unambiguously shown that yeast and human Hsp90 possess an adenine nucleotide binding site localized in the N-terminal part of the protein [13, 14]
In our previous reports we have established that Hsp90 rescues the WT p53 tumor suppressor protein activity at physiological temperature in a single chaperone reaction

Summary

EXPERIMENTAL PROCEDURES

Protein Purification—Human full-length Hsp90␤ isoform was cloned from human cDNA library and inserted into pBAD24 plasmid. Pull-down experiments were performed by incubation of 10 ␮l of the prepared affinity resin with 5 ␮g of the purified Hsp WT or mutant in the buffer: 25 mM Tris, pH 7.6, 50 mM KCl, 5 mM DTT, 10 mM MgCl2, 5 mM ATP. In Vitro p53 DNA Binding Assay—The p53 electrophoretic mobility shift assay (EMSA) was performed as described previously [25], with the modified reaction buffer composition: 25 mM Hepes, pH 7.5, 5% glycerol, 100 mM KCl, 10 mM MgCl2, and 3 mM DTT. Yeast and human Hsp90␣ protein variants were described previously to have impaired ATP cycling properties [14, 28]. D88N residue change, to abolish the binding of ATP to Hsp90␤ To verify these assumptions the WT and changed Hsp90␤ proteins were overproduced in E. coli and purified to homogeneity (Fig. 1A). They were properly folded as judged by CD spectrum and mostly dimeric, as judged by size-exclusion chromatography (data not shown)

All purified proteins were tested

RESULTS

DISCUSSION