Substrate recognition by the AAA+ chaperone ClpB

Schlieker, Christian; Weibezahn, Jimena; Patzelt, Holger; Tessarz, Peter; Strub, Christine; Zeth, Kornelius; Erbse, Annette H.; Schneider‐Mergener, Jens; Chin, Jason W.; Schultz, Peter G.; Bukau, Bernd; Mogk, Axel

doi:10.1038/nsmb787

Cited by 217 publications

(268 citation statements)

References 46 publications

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“…Thr213 in D1 of ClpB is positioned 31 Å away from an essential substrate-binding residue, Tyr251 located in an unstructured loop at the channel entrance. 5,11 A close proximity of the Walker-A Thr and the nucleotide c-phosphate (see Fig. 7) suggests that the threonine may participate in detecting the presence of c-phosphate and relaying that information to the substrate binding loop.…”

Section: Discussionmentioning

confidence: 96%

“…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%

“…5 The loops bind polypeptide substrates and are involved in their forced translocation. 11,13 ATP stabilizes the position of the D1 loop, whereas the loop retains its flexibility in the presence of ADP. 14 This observation is consistent with the ATP-induced high-affinity binding of substrates by ClpB and a low affinity of the ADP state 15 and implies a conformational linkage between the channel loop and the ATP-binding site.…”

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: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

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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“…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 .…”

mentioning

confidence: 99%

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

Doyle

Shorter

Żółkiewski

et al. 2007

Nat Struct Mol Biol

141

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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“…Instead of conventional carbon and nitrogen isotopes, the naturally abundant NMR-active nucleus of fluorine, 19 F can be incorporated into proteins. Fluorinated derivatives of phenylalanine (28), tyrosine (29) and lysine (30) have been successfully introduced using genetic code expansion [75][76][77]. Similarly, EPR measurements require the installation of stable radicals as spin-labels on proteins.…”

Section: Uaas With a Spin For Nmr And Eprmentioning

confidence: 99%

Rewiring translation – Genetic code expansion and its applications

Neumann

2012

FEBS Letters

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a b s t r a c tWith few minor variations, the genetic code is universal to all forms of life on our planet. It is difficult to imagine that one day organisms might exist that use an entirely different code to translate the information of their genome. Recent developments in the field of synthetic biology, however, have opened the gate to their creation. The genetic code of several organisms has been expanded by the heterologous expression of evolved aminoacyl-tRNA synthetase/tRNA CUA pairs that mediate the incorporation of unnatural amino acids in response to amber codons. These UAAs introduce exciting new features into proteins, such as spectroscopic probes, UV-inducible crosslinkers, and functional groups for bioorthogonal conjugations or posttranslational modifications. Orthogonal ribosomes provide a parallel translational machinery in Escherichia coli that has lost its evolutionary constraints. Evolved variants of these ribosomes translate amber or quadruplet codons with massively enhanced efficiency. Here, I review these recent developments emphasizing their tremendous potential to facilitate biochemical and cell biological studies.

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Substrate recognition by the AAA+ chaperone ClpB

Cited by 217 publications

References 46 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

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

Rewiring translation – Genetic code expansion and its applications

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