The ATPase Cycle of the Mitochondrial Hsp90 Analog Trap1

Abstract

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. 90-kDa heat shock protein (Hsp90)3 is a molecular chaperone highly conserved from bacteria to mammals. The dependence on Hsp90 apparently reflects the relative complexity of the organism. For example, bacteria lacking HtpG (the prokaryotic homolog of mammalian Hsp90) appear to be fully viable (1), whereas disruption of Hsp90 in eukaryotic organisms, including Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila, is lethal (2-5). The family of Hsp90-related molecular chaperones mediates protein folding, assembly, and stability of a variety of substrate proteins in vivo. In vitro, Hsp90 suppresses the aggregation of non-native proteins (6) and can also promote the refolding of substrates in cooperation with the Hsp70 system (7-10).In eukaryotic organisms, several members of the Hsp90 family can be found in different compartments such as the cytosol, endoplasmic reticulum, mitochondria and in chloroplasts (11). The organellar versions differ from their cytosolic counterparts in several aspects. The most striking is that they seem to lack specific co-chaperones. While Grp94, the Hsp90 homolog of the endoplasmic reticulum has evolved from its cytosolic counterpart, Trap1, the mitochondrial version, is a descendent of prokaryotic Hsp90 (11). Whether they share a general mechanism of ATP hydrolysis is not known. In this study, we focused on the biochemical and kinetic analysis of the Hsp90 homolog Trap1, which is localized in mitochondria (12-14). Originally, Trap1 was identified as a tumor necrosis factor receptor-associated protein (15). Trap1 is 31% identical to human Hsp90, 34% to Escherichia coli HtpG, 33% to yeast Hsp90, and 31% to Grp94. For the N-terminal ATP-binding domain alone, the identities are even higher. There are a few identified Trap1 substrates such as the retinoblastoma protein (16), the tumor suppressor EXT proteins (17) and Myc, which influences cell proliferation (18). Recently, an interaction partner of Trap1, which is locat...

TRAP1 regulation of mitochondrial life or death decision in cancer cells and mitochondria-targeted TRAP1 inhibitors

“…TRAP1 and Hsp90 share conserved domain architectures with high amino acid sequence similarity, molecular chaperone functions, and homo-dimeric quaternary structures (11)(12)(13)15). Apart from the structural similarity between them, TRAP1 has the additional mitochondrial targeting sequence at its N-terminus, which is cleaved off after mitochondrial translocation, and lacks the highly charged flexible region between the N and M domains of Hsp90 (11)(12)(13).…”

Cdc37-Hsp90 Complexes Are Responsive to Nucleotide-induced Conformational Changes and Binding of Further Cofactors

“…Indeed the degree of conservation between the Hsp90 proteins and their cofactors may argue for a conserved mechanism. In addition, all Hsp90 proteins studied to date share general aspects of the mechanism of nucleotideinduced transient N-terminal dimerization (14,16,17,63), but certainly the basic ATP turnover rates differ between 1 ATP/min in the yeast Hsp90 and 1 ATP/20 min in the human Hsp90s (5,29). Several cofactors were found to react differently in the yeast and human Hsp90 systems before.…”

“…The interaction of the N-terminal domains is initiated by a lid segment (ATP-lid), which closes over the nucleotide-binding pocket and subsequently exposes the N-terminal interaction surfaces (11)(12)(13)(14)(15). This model has been derived from extensive studies of yeast and human Hsp90 proteins and is believed to be conserved for all Hsp90s (14,16,17).…”

“…The interaction of the N-terminal domains is initiated by a lid segment (ATP-lid), which closes over the nucleotide-binding pocket and subsequently exposes the N-terminal interaction surfaces (11)(12)(13)(14)(15). This model has been derived from extensive studies of yeast and human Hsp90 proteins and is believed to be conserved for all Hsp90s (14,16,17).Co-chaperone regulation is an important feature of Hsp90 function, and a defined set of co-chaperones has been described for eukaryotic Hsp90 proteins (18). Inhibitory effects on the turnover rate of Hsp90 have been documented for the co-chaperones Sba1/p23 (9,19,20), Sti1/Hop (21, 22), and Cdc37 (23-25), whereas Aha1 and its homolog Hch1 are the only known activating cofactors to date (26 -28).…”

Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis

“…In multicellular eukaryotes, the HSP90 family includes the mitochondrial chaperone TRAP1 (TNF receptor-associated protein), which shares 50% sequence similarity with HSP90. Although TRAP1 binds and hydrolyzes ATP in an analogous manner to HSP90 (3), its cellular function is less well understood. Thus, although many HSP90-dependent proteins ("clients") and interacting cochaperones have been described (www.picard.ch/downloads/Hsp90interactors.pdf), the validated list of TRAP1-dependent clients is quite small and TRAP1-interacting cochaperones, if they exist, have yet to be identified (4).…”