The ATPase cycle of the mitochondrial Hsp90 analog Trap1.
Hsp90 is an ATP-dependent molecular chaperone whose mechanism is not yet understood in detail. Here, we present the first ATPase cycle for the mitochondrial member of the Hsp90 family called Trap1 (tumor necrosis factor receptor-associated protein 1). Using biochemical, thermodynamic, and rapid kinetic methods we dissected the kinetics of the nucleotide-regulated rearrangements between the open and the closed conformations. Surprisingly, upon ATP binding, Trap1 shifts predominantly to the closed conformation (70%), but, unlike cytosolic Hsp90 from yeast, this process is rather slow at 0.076 s(-1). Because reopening (0.034 s(-1)) is about ten times faster than hydrolysis (k(hyd) = 0.0039 s(-1)), which is the rate-limiting step, Trap1 is not able to commit ATP to hydrolysis. The proposed ATPase cycle was further scrutinized by a global fitting procedure that utilizes all relevant experimental data simultaneously. This analysis corroborates our model of a two-step binding mechanism of ATP followed by irreversible ATP hydrolysis and a one-step product (ADP) release.
Leskovar
Adriane
A
Department of Biomolecular Mechanisms, Max-Planck-Institute for Medical Research, Jahnstrasse 29, Heidelberg 69120, Germany.
Wegele
Harald
H
Werbeck
Nicolas D
ND
Buchner
Johannes
J
Reinstein
Jochen
J
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000378:metabolism; D002151:Calorimetry / Q000379:methods; D017259:Dimaprit / Q000031:analogs & derivatives / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D013816:Thermodynamics
D018841:HSP90 Heat-Shock Proteins; C092306:TRAP1 protein, human; D000244:Adenosine Diphosphate; C071989:APT; D000251:Adenosine Triphosphatases; D017259:Dimaprit