Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate

Abstract

Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90 N (TRAP1 N ) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis-bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding.TRAP1 | Hsp90 | molecular chaperone H eat-shock protein of 90 kDa (Hsp90) is a conserved ATPdependent molecular chaperone (1-4), which together with heat-shock protein of 70 kDa (Hsp70) (5-7) and a cohort of cochaperones (8-10), promotes the late-stage folding of Hsp90 client proteins (11). It is presumed that almost 400 different proteins, including a majority of signaling and tumor promoting proteins, depend on cytosolic Hsp90 for folding (12). Consequently, the ability to inactivate multiple oncogenic pathways simultaneously has made Hsp90 a major target for drug development (13), with several Hsp90 inhibitors currently undergoing clinical trials (14).Hsp90 chaperones display conformational plasticity in solution (2, 15, 16), with different adenine nucleotides either facilitating or stabilizing distinct Hsp90 dimer conformations (17-19). Interestingly, apo Hsp90 forms a wide-open, V-shaped dimer with the N domains separated by as much as 101 Å (18). This open conformation is markedly distinct from the intertwined, N-terminally closed dimer with ATP bound (20,21). Because the open-state dimer cannot signal the nucleotide status between neighboring subunits, an intermediate conformation preceding dimer closure must exist, which so far has remained elusive.Apart from cytosolic Hsp90s, Hsp90 homologs are found in the endoplasmic reticulum, chloroplasts, and mitochondria ( Fig. S1) (22). The tumor necrosis factor receptor-associated protein 1 (TRAP1) is the mitochondrial Hsp90 paralog, which prevents apoptosis and protects mitochondria against oxidative damage (23-25). TRAP1 is widely expressed in many tumors (24,26,27), but not in mitochondria of most normal tissues (24), benign prostatic hyperplasia (26), or highly proliferating, nontransformed cells (27). Notably, it was found that TRAP1 not only promotes neoplastic growth, but also confers tumorigenic potential on nontransformed cells (27), indicating a major rol...

The Chaperone TRAP1 As a Modulator of the Mitochondrial Adaptations in Cancer Cells

“…The ATPase cycle of TRAP1 has been investigated in detail (39, 41, 42) allowing to establish a model for its conformational cycle. Both TRAP1 protomers undergo concerted structural changes through rounds of ATP binding, hydrolysis, and release (39, 42), although it is unknown how ATP hydrolysis is coupled to client maturation.…”

“…Both TRAP1 protomers undergo concerted structural changes through rounds of ATP binding, hydrolysis, and release (39, 42), although it is unknown how ATP hydrolysis is coupled to client maturation. During the ATPase cycle, TRAP1 can adopt three distinct states (Figure 2): an open conformation, called the apo state; a closed conformation with an N-terminal strap/extension straddled between both protomers and a coiled-coil, intermediate conformation with the N-terminal domains in close physical proximity (40, 42). ATP binding induces a dramatic structural change in the chaperone configuration leading to the formation of a closed asymmetric conformation (39, 43).…”

“…Hydrolysis of the second ATP leads to the formation of the ADP state. The cycle eventually returns to the open conformation after ADP release (39–42). …”

S-nitrosylation affects TRAP1 structure and ATPase activity and modulates cell response to apoptotic stimuli

“…ATP binding and hydrolysis; a middle-domain (MD), that contains that contains part of the ATP-binding pocket [5], and the binding site for client proteins; a C-terminal domain (CTD) that constitutes the interface for homodimerization [6]. Differently form the other Hsp90 orthologs, TRAP1 lacks the linker domain between MD and CTD, and exhibits a N-terminal extension that acts as thermal regulator of its chaperone activity [7].The ATPase cycle of TRAP1 has been characterized in detail, with both protomers assisting the folding of the target proteins through structural modifications associated with repeated cycles of ATP binding, hydrolysis and release [2,8,9]. During the ATPase cycle, TRAP1 can be present under three different states: i) an open conformation (called apo); ii) a close conformation with the NTD placed between the two protomers; iii) an intermediate coiled-coil conformation with both NTD in close proximity.…”

“…The ATPase cycle of TRAP1 has been characterized in detail, with both protomers assisting the folding of the target proteins through structural modifications associated with repeated cycles of ATP binding, hydrolysis and release [2,8,9]. During the ATPase cycle, TRAP1 can be present under three different states: i) an open conformation (called apo); ii) a close conformation with the NTD placed between the two protomers; iii) an intermediate coiled-coil conformation with both NTD in close proximity.…”