Atypical AAA+ Subunit Packing Creates an Expanded Cavity for Disaggregation by the Protein-Remodeling Factor Hsp104

Wendler, Petra; Shorter, James; Plisson, Célia; Cashikar, Anil G.; Lindquist, Susan; Saibil, Helen R.

doi:10.1016/j.cell.2007.10.047

Cited by 113 publications

(198 citation statements)

References 59 publications

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“…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 cooperated with the eukaryotic Hsp70/40 system and vice versa (7,27). The role of the M-domain in mediating DnaKJ/GrpE interaction is consistent with the M-domain being on the outside of the ClpB hexamer (5,8), but incompatible with the previously proposed structure of yeast Hsp104 (28,29), which placed the M-domains on the interior or intercalated between subunits.…”

supporting

confidence: 64%

Structural basis for intersubunit signaling in a protein disaggregating machine

Biter

Lee

Sung

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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...

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supporting

confidence: 64%

Structural basis for intersubunit signaling in a protein disaggregating machine

Biter

Lee

Sung

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…We first investigated whether these methods could be applied to macromolecules in extracts from cells expressing chromosomally green fluorescent protein (GFP)-tagged proteasome subunit (Pre6) 14,21 , Hsp104, or ribosome subunit Rpl19a (Fig. 2) [31][32][33] . All of the FCF curves fit well to a one-component diffusion model (Fig.…”

Section: Resultsmentioning

confidence: 99%

Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

et al. 2014

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The 26S proteasome is a 2.5-MDa multisubunit protease complex that degrades polyubiquitylated proteins. Although its functions and structure have been extensively characterized, little is known about its dynamics in living cells. Here, we investigate the absolute concentration, spatio-temporal dynamics and complex formation of the proteasome in living cells using fluorescence correlation spectroscopy. We find that the 26S proteasome complex is highly mobile, and that almost all proteasome subunits throughout the cell are stably incorporated into 26S proteasomes. The interaction between 19S and 20S particles is stable even in an importin-a mutant, suggesting that the 26S proteasome is assembled in the cytoplasm. Furthermore, a genetically stabilized 26S proteasome mutant is able to enter the nucleus. These results suggest that the 26S proteasome completes its assembly process in the cytoplasm and translocates into the nucleus through the nuclear pore complex as a holoenzyme.

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“…2,3 Hsp104 and its prokaryotic homologue ClpB are hexameric AAA + ATPases with two nucleotide-binding domains (NBDs) per subunit that couple asymmetric cycles of ATP hydrolysis to the disaggregation of their polypeptide substrates. The substrate binding of ClpB/Hsp104 was shown to be dependent on the ATP state (Scheme 1a); accordingly, reduced or impaired ATP hydrolysis, as in the case of the ATPase mutant E285Q/E687Q (Hsp104 TRAP ), increases the affinity towards polypeptide substrates 5,7,8 (Scheme 1b). The processing of polypeptides by wild-type ClpB/Hsp104 results in the unfolding of non-native structures and separation of single polypeptides from aggregates, thus facilitating the refolding of proteins.…”

Section: Introductionmentioning

confidence: 99%

Hsp104 Targets Multiple Intermediates on the Amyloid Pathway and Suppresses the Seeding Capacity of Aβ Fibrils and Protofibrils

Arimon

Grimminger

Sanz

et al. 2008

Journal of Molecular Biology

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The heat shock protein Hsp104 has been reported to possess the ability to modulate protein aggregation and toxicity and to "catalyze" the disaggregation and recovery of protein aggregates, including amyloid fibrils, in yeast, Escherichia coli, mammalian cell cultures, and animal models of Huntington's disease and Parkinson's disease. To provide mechanistic insight into the molecular mechanisms by which Hsp104 modulates aggregation and fibrillogenesis, the effect of Hsp104 on the fibrillogenesis of amyloid beta (Aβ) was investigated by characterizing its ability to interfere with oligomerization and fibrillogenesis of different species along the amyloid-formation pathway of Aβ. To probe the disaggregation activity of Hsp104, its ability to dissociate preformed protofibrillar and fibrillar aggregates of Aβ was assessed in the presence and in the absence of ATP. Our results show that Hsp104 inhibits the fibrillization of monomeric and protofibrillar forms of Aβ in a concentration-dependent but ATP-independent manner. Inhibition of Aβ fibrillization by Hsp104 is observable up to Hsp104/Aβ stoichiometric ratios of 1:1000, suggesting a preferential interaction of Hsp104 with aggregation intermediates (e.g., oligomers, protofibrils, small fibrils) on the pathway of Aβ amyloid formation. This hypothesis is consistent with our observations that Hsp104 (i) interacts with Aβ protofibrils, (ii) inhibits conversion of protofibrils into amyloid fibrils, (iii) arrests fibril elongation and reassembly, and (iv) abolishes the capacity of protofibrils and sonicated fibrils to seed the fibrillization of monomeric Aβ. Together, these findings suggest that the strong inhibition of Aβ fibrillization by Hsp104 is mediated by its ability to act at different stages and target multiple intermediates on the pathway to amyloid formation.

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Atypical AAA+ Subunit Packing Creates an Expanded Cavity for Disaggregation by the Protein-Remodeling Factor Hsp104

Cited by 113 publications

References 59 publications

Structural basis for intersubunit signaling in a protein disaggregating machine

Structural basis for intersubunit signaling in a protein disaggregating machine

Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

Hsp104 Targets Multiple Intermediates on the Amyloid Pathway and Suppresses the Seeding Capacity of Aβ Fibrils and Protofibrils

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