Coupling ATP utilization to protein remodeling by ClpB, a hexameric AAA+ protein

Hoskins, Joel R.; Doyle, Shannon M.; Wickner, Sue

doi:10.1073/pnas.0911937106

Cited by 49 publications

(73 citation statements)

References 39 publications

(48 reference statements)

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“…S1) [16]. We also compare data presented herein with recently published results obtained with mixing experiments, in which different amounts of homohexamers of wt ClpB and some of the protein mutants used in this study are mixed before the experiment [16,18].…”

Section: Resultssupporting

confidence: 66%

“…A recent study with TClpB has used this property to show that insertion of an inactive subunit within the hexamer impairs its chaperone activity [16]. Using the same mixing approach, it has been recently shown that ClpB can modify the mechanism of ATP utilization depending on the substrate and the presence of the DnaK system [18]. While in the absence of DnaK the protein can remodel the substrate with approximately three active protomers per hexamer, in the presence of DnaK the characteristics of the active hexamer have not been defined so accurately.…”

Section: Introductionmentioning

confidence: 99%

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Nucleotide utilization requirements that render ClpB active as a chaperone

Castillo¹,

Fernández-Higuero²,

Pérez-Acebrón³

et al. 2010

FEBS Letters

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a b s t r a c tClpB is a member of the AAA+ superfamily that forms a ring-shaped homohexamer. Each protomer contains two nucleotide binding domains arranged in two rings that hydrolyze ATP. We extend here previous studies on ClpB nucleotide utilization requirements by using an experimental approach that maximizes random incorporation of different subunits into the protein hexamer. Incorporation of one subunit unable to bind or hydrolyze ATP knocks down the chaperone activity, while the wt hexamer can accommodate two mutant subunits that hydrolyze ATP in only one protein ring. Four subunits seem to build the functional cooperative unit, provided that one of the protein rings contains active nucleotide binding sites.

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Section: Resultssupporting

confidence: 66%

Section: Introductionmentioning

confidence: 99%

Nucleotide utilization requirements that render ClpB active as a chaperone

Castillo¹,

Fernández-Higuero²,

Pérez-Acebrón³

et al. 2010

FEBS Letters

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show abstract

“…As anticipated, many of our substrates were not reactivated, including chemically denatured FFL, which requires both Hsp70 and Hsp40 for protein refolding (42,43). Strikingly, we found that both aggregated β-gal and EGFP, which do not require Hsp70/40 for reactivation (6,31,44), could be rescued by Hsp104 together with full-length Hsp70 (Fig. 5A and Fig.…”

Section: Resultsmentioning

confidence: 54%

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

Lee¹,

Kim²,

Biter³

et al. 2013

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

102

111

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Heat shock protein (Hsp) 104 is a ring-forming, protein-remodeling machine that harnesses the energy of ATP binding and hydrolysis to drive protein disaggregation. Although Hsp104 is an active ATPase, the recovery of functional protein requires the speciesspecific cooperation of the Hsp70 system. However, like Hsp104, Hsp70 is an active ATPase, which recognizes aggregated and aggregation-prone proteins, making it difficult to differentiate the mechanistic roles of Hsp104 and Hsp70 during protein disaggregation. Mapping the Hsp70-binding sites in yeast Hsp104 using peptide array technology and photo-cross-linking revealed a striking conservation of the primary Hsp70-binding motifs on the Hsp104 middle-domain across species, despite lack of sequence identity. Remarkably, inserting a Strep-Tactin binding motif at the spatially conserved Hsp70-binding site elicits the Hsp104 protein disaggregating activity that now depends on Strep-Tactin but no longer requires Hsp70/40. Consistent with a Strep-Tactin-dependent activation step, we found that full-length Hsp70 on its own could activate the Hsp104 hexamer by promoting intersubunit coordination, suggesting that Hsp70 is an activator of the Hsp104 motor.ClpB | DnaK | Hsp100 | molecular chaperone

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“…We propose that this signaling network is crucial to sense the nucleotide state in the adjacent subunit and to reset the nucleotide cycle in the ClpB ring following ATP hydrolysis. The existence of an ISS network that regulates ATP hydrolysis in diverse AAA+ ring complexes is also consistent with a sequential ATP-hydrolysis mechanism proposed for ClpB (45,46) and Hsp104 (47), with four out of six subunits in the ClpB homohexamer occupied by nucleotides at any one time (46). This model is similar to the staircase mechanism proposed for the T7 gene 4 ring helicase (48), and is consistent with the nucleotide occupancy observed in the crystal structure of an engineered, covalently linked ClpX hexamer (11).…”

Section: Discussionmentioning

confidence: 51%

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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Coupling ATP utilization to protein remodeling by ClpB, a hexameric AAA+ protein

Cited by 49 publications

References 39 publications

Nucleotide utilization requirements that render ClpB active as a chaperone

Nucleotide utilization requirements that render ClpB active as a chaperone

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

Structural basis for intersubunit signaling in a protein disaggregating machine

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