Evidence for iterative ratcheting of receptor-bound hsp70 between its ATP and ADP conformations during assembly of glucocorticoid receptor.hsp90 heterocomplexes.
hsp90 and hsp70 are essential components of a five-protein system, including also the nonessential cochaperones Hop, hsp40, and p23, that assembles glucocorticoid receptor (GR).hsp90 heterocomplexes and causes the simultaneous opening of the steroid binding pocket to access by steroid. The first event in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR, which primes the receptor for subsequent ATP-dependent activation by hsp90 [Morishima, Y., Murphy, P. J. M., Li, D. P., Sanchez, E. R., and Pratt, W. B. (2000) J. Biol. Chem. 275, 18054-18060]. Here, we demonstrate that, during the priming step, ATP-bound hsp70 is converted to GR-bound hsp70 that is approximately 1/3 in the ADP- and approximately 2/3 in the ATP-dependent conformation. In the second step, hsp90, which is provided in the non-nucleotide-bound state, is converted to GR-bound hsp90 in the ATP-dependent conformation. The ATPase activity of hsp70 is K(+)-dependent, and the priming step is K(+)-dependent. Surprisingly, the subsequent hsp90-dependent step, which is rate-limiting for receptor activation, is also potassium-dependent. This suggests that GR-bound hsp70 is also converted from the ATP-dependent to the ADP-dependent conformation while it cooperates with hsp90 to activate steroid binding activity. Because the priming step requires both sustained high levels of ATP and YDJ-1 for optimal activity and because both steps require potassium, we predict that receptor-bound hsp70 undergoes iterative ratcheting between its ATP- and ADP-dependent conformations in opening the hydrophobic steroid binding pocket.
Morishima
Y
Y
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Kanelakis
K C
KC
Murphy
P J
PJ
Shewach
D S
DS
Pratt
W B
WB
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D019746:Intramolecular Oxidoreductases; D046911:Macromolecular Substances; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D000071876:Prostaglandin-E Synthases; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D011869:Radioligand Assay; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000378:metabolism*; D018411:Spodoptera
C494647:DNAJB1 protein, human; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D010750:Phosphoproteins; D011965:Receptors, Glucocorticoid; C079373:STIP1 protein, human; C489791:Stip1 protein, mouse; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D019746:Intramolecular Oxidoreductases; C490849:PTGES3 protein, human; D000071876:Prostaglandin-E Synthases