<i>Escherichia coli</i> ClpB is a non-processive polypeptide translocase

Li, Tao; Weaver, Clarissa L.; Lin, Jeffrey; Duran, Elizabeth C.; Miller, Justin M.; Lucius, Aaron L.

doi:10.1042/bj20141457

Cited by 38 publications

(75 citation statements)

References 60 publications

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“…Indeed, the ClpA 50 and ClpB 25 protomers form a continuous right-handed spiral in the crystal. However the conformations are overall different compared to our Hsp104 cryo-EM structure and both display mechanistic differences; ClpA functions in conjunction with a bound ClpP protease 51 , while ClpB is proposed to operate via non-processive translocation events 35,46 , thus these complexes may adopt different hexamer arrangements in solution.…”

Section: Discussionmentioning

confidence: 67%

“…Nonetheless, the staggered arrangement of binding surfaces explains how unidirectional threading can confer more powerful unfolding. While the translocation step size is unknown for Hsp104, it is expected to be just a few amino acids for ClpB 46 , and has been measured to be 5–8 amino acids for ClpX 47,48 and even smaller and more regular for ClpA 49 . Thus, with the 15–20 Å distance between pore loops we identify, interaction sites spanning a small number of amino acids along a polypeptide can be transferred stepwise from one protomer to the next during ATP hydrolysis, facilitating processive translocation through the channel.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation

Yokom

Gates

Jackrel

et al. 2016

Nat Struct Mol Biol

103

206

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ClpB and Hsp104 are conserved AAA+ protein disaggregases that promote survival during cellular stress. Hsp104 acts on amyloids, supporting prion propagation in yeast, and can solubilize toxic oligomers connected to neurodegenerative diseases. A definitive structural mechanism, however, has remained elusive. We have determined the cryo-EM structure of Hsp104 in the ATP state, revealing a near-helical hexamer architecture that coordinates the mechanical power of the twelve AAA+ domains for disaggregation. An unprecedented heteromeric AAA+ interaction defines an asymmetric seam in an apparent catalytic arrangement that aligns the domains in a two-turn spiral. N-terminal domains interact to form a broad channel entrance for substrate engagement and Hsp70 interaction. Middle-domain helices bridge adjacent protomers across the nucleotide pocket, explaining roles in hydrolysis and disaggregation. Remarkably, substrate-binding pore loops line the channel in a continuous spiral that appears optimized for substrate transfer across the AAA+ domains, establishing a directional path for polypeptide translocation.

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Section: Discussionmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation

Yokom

Gates

Jackrel

et al. 2016

Nat Struct Mol Biol

103

206

View full text Add to dashboard Cite

show abstract

“…Structural characterization of NSF-SNARE complexes by cryo-electron microscopy (cryo-EM) 104 and single-molecule experiments suggest a spring-loaded mechanism of disassembly 105 . ClpB may remodel some bacterial substrates by a similar mechanism, as a recent study suggests that it dissociates from an unfolded substrate after only one or two translocation steps 106 .…”

Section: Aaa+ Remodelling Machinesmentioning

confidence: 97%

Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines

2015

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Preface To maintain protein homeostasis, AAA+ proteolytic machines degrade damaged and unneeded proteins in bacteria, archaea and eukaryotes. This process involves ATP-dependent unfolding of a target protein and subsequent translocation into a self-compartmentalized proteolytic chamber. Related AAA+ enzymes also disaggregate and remodel proteins. Recent structural and biochemical studies, in combination with direct visualization of unfolding and translocation in single-molecule experiments, have illuminated molecular mechanisms and suggest how remodelling of macromolecular complexes by AAA+ enzymes could occur without global denaturation. In this Review, we discuss the structural and mechanistic features of AAA+ proteases and remodeling machines, focusing on bacterial ClpXP and ClpX as paradigms. We also consider the potential of these enzymes as antibacterial targets and outline future challenges for the field.

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“…Protein disaggregation is driven by Hsp104 coupling ATP hydrolysis to the partial or complete translocation of substrate across the central channel to solution via interaction with conserved tyrosine-bearing pore loops in NBD1 and NBD2 [33-37, 41]. However, recent studies suggest that ClpB is a non-processive translocase, which takes only 1-2 translocation steps prior to releasing substrate, which appears insufficient for complete translocation across the central channel [121]. Thus, ClpB may dissociate protein aggregates by pulling and releasing exposed tails or loops [121].…”

Section: Hsp104 Structure and Mechanismmentioning

confidence: 99%

“…However, recent studies suggest that ClpB is a non-processive translocase, which takes only 1-2 translocation steps prior to releasing substrate, which appears insufficient for complete translocation across the central channel [121]. Thus, ClpB may dissociate protein aggregates by pulling and releasing exposed tails or loops [121]. In light of these studies [121], it will be important to determine the processivity of Hsp104.…”

Section: Hsp104 Structure and Mechanismmentioning

confidence: 99%

Mechanistic and Structural Insights into the Prion-Disaggregase Activity of Hsp104

Sweeny

Shorter

2016

Journal of Molecular Biology

108

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Hsp104 is a dynamic ring-translocase and hexameric AAA+ protein found in yeast, which couples ATP hydrolysis to disassembly and reactivation of proteins trapped in soluble preamyloid oligomers, disordered protein aggregates, and stable amyloid or prion conformers. Here, we highlight advances in our structural understanding of Hsp104 and how Hsp104 deconstructs Sup35 prions. Although the atomic structure of Hsp104 hexamers remains uncertain, volumetric reconstruction of Hsp104 hexamers in ATPγS, ADP-AlFx (ATP hydrolysis transition state mimic), and ADP via small-angle x-ray scattering has revealed a peristaltic pumping motion upon ATP hydrolysis. This pumping motion likely drives directional substrate translocation across the central Hsp104 channel. Hsp104 initially engages Sup35 prions immediately C-terminal to their cross-β structure. Directional pulling by Hsp104 then resolves N-terminal cross-β structure in a stepwise manner. First, Hsp104 fragments the prion. Second, Hsp104 unfolds cross-β structure. Third, Hsp104 releases soluble Sup35. Deletion of the Hsp104 N-terminal domain (NTD) yields a hypomorphic disaggregase, Hsp104ΔN, with an altered pumping mechanism. Hsp104ΔN fragments Sup35 prions without unfolding cross-β structure or releasing soluble Sup35. Moreover, Hsp104ΔN activity cannot be enhanced by mutations in the middle domain that potentiate disaggregase activity. Thus, the NTD is critical for the full repertoire of Hsp104 activities.

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Escherichia coli ClpB is a non-processive polypeptide translocase

Abstract: Here we show that ClpB is a non-processive translocase that takes, at most, two steps on the polypeptide backbone before dissociation. These findings indicate that ClpB is not likely to translocate polypeptide through its axial channel as previously concluded.

Cited by 38 publications

References 60 publications

Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation

Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation

Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines

Mechanistic and Structural Insights into the Prion-Disaggregase Activity of Hsp104

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