ClpB is a ring-forming, ATP-dependent protein disaggregase that cooperates with the cognate Hsp70 system to recover functional protein from aggregates. How ClpB harnesses the energy of ATP binding and hydrolysis to facilitate the mechanical unfolding of previously aggregated, stress-damaged proteins remains unclear.Here, we present crystal structures of the ClpB D2 domain in the nucleotide-bound and -free states, and the fitted cryoEM structure of the D2 hexamer ring, which provide a structural understanding of the ATP power stroke that drives protein translocation through the ClpB hexamer. We demonstrate that the conformation of the substrate-translocating pore loop is coupled to the nucleotide state of the cis subunit, which is transmitted to the neighboring subunit via a conserved but structurally distinct intersubunit-signaling pathway common to diverse AAA+ machines. Furthermore, we found that an engineered, disulfide cross-linked ClpB hexamer is fully functional biochemically, suggesting that ClpB deoligomerization is not required for protein disaggregation.ATPase | chaperone | Hsp100 | protein unfoldase C lpB is an ATP-dependent protein-remodeling machine that has the remarkable ability to rescue stress-damaged proteins from a previously aggregated state. As the major protein disaggregase in cells, bacterial ClpB and its yeast (Hsp104) and plant (Hsp101) homologs are essential for thermotolerance development (1-3), and for cell survival from acute stress conditions (4).At the molecular level, ClpB is a multidomain protein composed of two tandem Walker-type ATP-binding domains (AAA+ domains), termed D1 and D2, which drive ClpB's chaperone activity. The D1 domain features the ClpB-specific M-domain, which forms a long coiled-coil (5) and is essential for protein disaggregation (6, 7). Like other type II AAA+ ATPases, ClpB forms a double-ring structure, with six copies of the D1 and D2 domains each making up a homohexamer ring (5,8). Although ClpB shares similar quaternary structure with ClpA (9), ClpC (10), and the single-ring ClpX (11) and HslU (12, 13) AAA+ ATPases, which function as the protein unfoldase components of energy-dependent proteases, ClpB does not associate with a chambered peptidase to degrade proteins. Instead, ClpB cooperates with the cognate Hsp70 system (DnaKJ/GrpE) in a species-specific manner (14, 15) to recover functional protein from aggregates (16-18).The prevailing model suggests that DnaKJ/GrpE targets the ClpB motor activity to aggregates (19,20), which is consistent with an upstream role of the DnaK system in protein disaggregation (21-23). Once targeted, ClpB disaggregates protein aggregates by extracting unfolded polypeptides (24) and threading them through the ClpB hexamer ring (21,25). In support of a direct ClpB-DnaKJ/GrpE interaction, it was reported that ClpB interacts with DnaK via the ClpB M-domain (15,26). Notably, replacing the M-domain of bacterial ClpB with that of its yeast homolog Hsp104 switched the species specificity of the bichaperone system so that ClpB now c...

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...

SUMMARY Hsp104 is a ring-forming, ATP-driven molecular machine that recovers functional protein from both stress-denatured and amyloid-forming aggregates. Although Hsp104 shares a common architecture with Clp/Hsp100 protein unfoldases, different and seemingly conflicting 3D structures have been reported. Examining the structure of Hsp104 poses considerable challenges because Hsp104 readily hydrolyzes ATP, whereas ATP analogs can be slowly turned over and are often contaminated with other nucleotide species. Here, we present the single-particle electron cryo-microscopy (cryo-EM) structures of a catalytically inactive Hsp104 variant (Hsp104DWB) in the ATP-bound state determined between 7.7 Å and 9.3 Å resolution. Surprisingly, we observe that the Hsp104DWB hexamer adopts distinct ring conformations (closed, extended, and open) despite being in the same nucleotide state. The latter underscores the structural plasticity of Hsp104 in solution, with different conformations stabilized by nucleotide binding. Our findings suggest that, in addition to ATP hydrolysis-driven conformational changes, Hsp104 uses stochastic motions to translocate unfolded polypeptides.

The ring-forming Hsp104 ATPase cooperates with Hsp70 and Hsp40 molecular chaperones to rescue stress-damaged proteins from both amorphous and amyloid-forming aggregates. The ability to do so relies upon pore loops present in the first ATP-binding domain (AAA-1; loop-1 and loop-2 ) and in the second ATP-binding domain (AAA-2; loop-3) of Hsp104, which face the protein translocating channel and couple ATP-driven changes in pore loop conformation to substrate translocation. A hallmark of loop-1 and loop-3 is an invariable and mutational sensitive aromatic amino acid (Tyr257 and Tyr662) involved in substrate binding. However, the role of conserved aliphatic residues (Lys256, Lys258, and Val663) flanking the pore loop tyrosines, and the function of loop-2 in protein disaggregation has not been investigated. Here we present the crystal structure of an N-terminal fragment of Saccharomyces cerevisiae Hsp104 exhibiting molecular interactions involving both AAA-1 pore loops, which resemble contacts with bound substrate. Corroborated by biochemical experiments and functional studies in yeast, we show that aliphatic residues flanking Tyr257 and Tyr662 are equally important for substrate interaction, and abolish Hsp104 function when mutated to glycine. Unexpectedly, we find that loop-2 is sensitive to aspartate substitutions that impair Hsp104 function and abolish protein disaggregation when loop-2 is replaced by four aspartate residues. Our observations suggest that Hsp104 pore loops have non-overlapping functions in protein disaggregation and together coordinate substrate binding, unfolding, and translocation through the Hsp104 hexamer.

Hsp104 is a double ring-forming AAA+ ATPase, which harnesses the energy of ATP binding and hydrolysis to rescue proteins from a previously aggregated state. Like other AAA+ machines, Hsp104 features conserved cis- and trans-acting elements, which are hallmarks of AAA+ members and are essential to Hsp104 function. Despite these similarities, it was recently proposed that Hsp104 is an atypical AAA+ ATPase, which markedly differs in 3D structure from other AAA+ machines. Consequently, it was proposed that arginines found in the non-conserved M-domain, but not the predicted Arg-fingers, serve the role of the critical trans-acting element in Hsp104. While the structural discrepancy has been resolved, the role of the Arg-finger residues in Hsp104 remains controversial. Here, we exploited the ability of Hsp104 variants featuring mutations in one ring to retain ATPase and chaperone activities, to elucidate the functional role of the predicted Arg-finger residues. We found that the evolutionarily conserved Arg-fingers are absolutely essential for ATP hydrolysis but are dispensable for hexamer assembly in Hsp104. On the other hand, M-domain arginines are not strictly required for ATP hydrolysis and affect the ATPase and chaperone activities in a complex manner. Our results confirm that Hsp104 is not an atypical AAA+ ATPase, and uses conserved structural elements common to diverse AAA+ machines to drive the mechanical unfolding of aggregated proteins.