Thermotolerance Requires Refolding of Aggregated Proteins by Substrate Translocation through the Central Pore of ClpB

Weibezahn, Jimena; Tessarz, Peter; Schlieker, Christian; Zahn, Regina; Maglica, Željka; Lee, Sukyeong; Zentgraf, Hanswalter; Weber‐Ban, Eilika; Dougan, David A.; Tsai, Francis T.F.; Mogk, Axel; Bukau, Bernd

doi:10.1016/j.cell.2004.11.027

Cited by 443 publications

(535 citation statements)

References 41 publications

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“…The mechanism of ATPase activation by casein or polylysine is not known, but it is accepted that the ATPase acceleration accompanies translocation of the soluble pseudosubstrates through the channel of ClpB. 9 Thus, our results suggest that the translocation machinery may remain functional in the ClpB variants with the noncanonical Walker A motif.…”

Section: Resultsmentioning

confidence: 64%

“…[6][7][8] The ClpB activity is linked to extraction of single polypeptides from aggregated particles and their forced unfolding by translocation through a narrow channel located at the center of the hexameric ring. 9 The substrate translocation by ClpB is driven by ATP hydrolysis and is analogous to the degradationpreceding unfolding/translocation of substrates by the proteasome and other ATP-dependent proteases. 10 Aggregated substrates bind to D1 and to the N-terminal domain of ClpB, 11,12 become translocated through the channel from D1 to D2, then exit the ClpB oligomer and their further folding in solution may be assisted by other chaperones.…”

Section: Introductionmentioning

confidence: 99%

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Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Nagy

et al. 2009

Protein Science

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Hexameric AAA1 ATPases induce conformational changes in a variety of macromolecules. AAA1 structures contain the nucleotide-binding P-loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA1 sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA1 ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker-A Thr with Asn in ClpB, a bacterial AAA1 chaperone which reactivates aggregated proteins. We found that the T-to-N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP-bound conformation and the high-affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker-A Thr in sensing the nucleotide's c-phosphate and in maintaining an allosteric linkage between the P-loop and the aggregate binding site of ClpB. We postulate that AAA1 ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate-remodeling activity.

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Section: Resultsmentioning

confidence: 64%

Section: Introductionmentioning

confidence: 99%

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Nagy

et al. 2009

Protein Science

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“…All Hsp100s self-assembly into ring-shaped structures and conformational changes are triggered by ATP binding and hydrolysis, which changes the positions of bound substrates relative to each other, followed by the translocation of the polypeptide through the central channel during aggregate reactivation (Weibezahn et al 2004, Zolkiewski 2006. By using E. coli ClpB as a model, Schlieker et al (2004) showed that the disaggregation mechanism involves continuous extraction of unfolded proteins from the aggregate that are solubilized into the central channel in a process called "threading".…”

mentioning

confidence: 99%

“…Likewise, the hexameric structure of ClpB from Thermus thermophilus (Fig. 1b) is crucial for its activity because it provides multiple sites for polypeptide substrate binding, which are translocated through its central channel during aggregate reactivation (Schirmer et al 1996, Lee et al 2003, Schlieker et al 2004, Weibezahn et al 2004, Zolkiewski 2006, Barends et al 2010, Mayer 2010, Zolkiewski et al 2012, Doyle et al 2013). The solubilized protein can then be refolded either spontaneously, or by a chaperone network independent of ClpB.…”

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confidence: 99%

Disaggregases, molecular chaperones that resolubilize protein aggregates

Mokry

Abrahão

Ramos

2015

An. Acad. Bras. Ciênc.

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The process of folding is a seminal event in the life of a protein, as it is essential for proper protein function and therefore cell physiology. Inappropriate folding, or misfolding, can not only lead to loss of function, but also to the formation of protein aggregates, an insoluble association of polypeptides that harm cell physiology, either by themselves or in the process of formation. Several biological processes have evolved to prevent and eliminate the existence of non-functional and amyloidogenic aggregates, as they are associated with several human pathologies. Molecular chaperones and heat shock proteins are specialized in controlling the quality of the proteins in the cell, specifically by aiding proper folding, and dissolution and clearance of already formed protein aggregates. The latter is a function of disaggregases, mainly represented by the ClpB/Hsp104 subfamily of molecular chaperones, that are ubiquitous in all organisms but, surprisingly, have no orthologs in the cytosol of metazoan cells. This review aims to describe the characteristics of disaggregases and to discuss the function of yeast Hsp104, a disaggregase that is also involved in prion propagation and inheritance.

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“…ATP is bound at the interface between adjacent subunits 12 and hexamerization is stabilized by nucleotide 13, 14 . Substrate binding occurs when the hexamer is in its ATP-bound conformation and conserved pore residues may contact substrates directly [15][16][17] . The N-and C-terminal domains may also help engage substrates and co-factors 18,19 .…”

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confidence: 99%

Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity

Doyle

Shorter

Żółkiewski

et al. 2007

Nat Struct Mol Biol

147

229

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Two members of the AAA+ superfamily, ClpB and Hsp104, collaborate with Hsp70 and Hsp40 to rescue aggregated proteins. However, the mechanisms that elicit and underlie their proteinremodeling activities remain unclear. We report that for both Hsp104 and ClpB, mixtures of ATP and ATPγS unexpectedly unleash activation, disaggregation, and unfolding activities independent of co-chaperones. Mutations reveal how remodeling activities are elicited by impaired hydrolysis at individual nucleotide binding domains. However, for some substrates, mixtures of ATP and ATPγS abolish remodeling, while for others ATP binding without hydrolysis is sufficient. Remodeling of different substrates necessitates a diverse balance of polypeptide holding (which requires ATP binding but not hydrolysis) and unfolding (which requires ATP hydrolysis). We suggest that this versatility in reaction mechanism enables ClpB and Hsp104 to reactivate the entire aggregated proteome after stress, and enables Hsp104 to control prion inheritance.Life demands that members of the AAA+ ATPase superfamily (ATPases associated with various cellular activities) couple energy from ATP hydrolysis to the remodeling of a bewildering array of macromolecular structures, that range from protein to DNA and RNA 1, 2 . Typically, eukaryotic genomes encode 50-80 family members 1 , each of which occupies specific niches that require specialized modes of substrate selection and regulation. The extraordinary adaptive radiation of AAA+ proteins to function in a multitude of cellular reactions illustrates the versatility of their structurally conserved AAA+ domain. Subunits containing AAA+ domains assemble into oligomeric rings, and ATP binds at the interface between adjacent protomers 1, 2 . AAA+ oligomers undergo considerable conformational changes during ATP binding and hydrolysis, although how these events are regulated and transduced into productive substrate remodeling remains largely enigmatic. Furthermore, it remains unanswered whether individual AAA+ family members rely on a common reaction mechanism to remodel various macromolecular clients. It is also unclear whether different AAA+ members have evolved distinct methods to engage and restructure substrates, or if individual proteins can switch between distinct reaction mechanisms for different substrates.Two members of the AAA+ superfamily separated by ~2 billion years of evolution 3 , yeast Hsp104, and its E. coli homolog, ClpB, allow cell survival after exposure to extreme environmental stress 4-7 . They function to dissolve and renature thousands of diverse 5 Correspondence: Sue Wickner,

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Thermotolerance Requires Refolding of Aggregated Proteins by Substrate Translocation through the Central Pore of ClpB

Cited by 443 publications

References 41 publications

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Disaggregases, molecular chaperones that resolubilize protein aggregates

Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity

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