ATP binding to Hsp90 is sufficient for effective chaperoning of p53 protein.
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.
Walerych
Dawid
D
International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.
Gutkowska
Malgorzata
M
Klejman
Marcin P
MP
Wawrzynow
Bartosz
B
Tracz
Zuzanna
Z
Wiech
Milena
M
Zylicz
Maciej
M
Zylicz
Alicja
A
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002460:Cell Line; D005786:Gene Expression Regulation; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D020033:Protein Isoforms; D016159:Tumor Suppressor Protein p53; D000255:Adenosine Triphosphate