M Domains Couple the ClpB Threading Motor with the DnaK Chaperone Activity

Haslberger, Tobias; Weibezahn, Jimena; Zahn, Regina; Lee, Sukyeong; Tsai, Francis T.F.; Bukau, Bernd; Mogk, Axel

doi:10.1016/j.molcel.2006.11.008

Cited by 156 publications

(213 citation statements)

References 34 publications

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“…The potential crowbar function is also unnecessary to disaggregate aggregated GFP, another reaction that both chaperones can separately carry out. The possibility that the middle domain may mediate communication between NBD-1 and NBD-2 (20) or between ClpB and DnaK seems more plausible (21). Regardless of the precise mechanism, our results show that the two systems work synergistically in protein disaggregation, with the DnaK system very likely acting concomitantly with ClpB.…”

Section: Discussionmentioning

confidence: 65%

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Collaboration between the ClpB AAA+ remodeling protein and the DnaK chaperone system

Doyle

Hoskins

Wickner³

2007

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

104

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ClpB and Hsp104, members of the AAA؉ superfamily of proteins, protect cells from the devastating effects of protein inactivation and aggregation that arise after extreme heat stress. They exist as a hexameric ring and contain two nucleotide-binding sites per monomer. ClpB and Hsp104 are able to dissolve protein aggregates in conjunction with the DnaK/Hsp70 chaperone system, although the roles of the individual chaperones in disaggregation are not well understood. In the absence of the DnaK/Hsp70 system, ClpB and Hsp104 alone are able to perform protein remodeling when their ATPase activity is asymmetrically slowed either by providing a mixture of ATP and ATP␥S, a nonphysiological and slowly hydrolyzed ATP analog, or by inactivating one of the two nucleotide-binding domains by mutation. To gain insight into the roles of ClpB and the DnaK system in protein remodeling, we tested whether there was a further stimulation by the DnaK chaperone system under conditions that elicited remodeling activity by ClpB alone. Our results demonstrate that ClpB and the DnaK system act synergistically to remodel proteins and dissolve aggregates. The results further show that ATP is required and that both nucleotidebinding sites of ClpB must be able to hydrolyze ATP to permit functional collaboration between ClpB and the DnaK system.S evere cellular stress results in extensive protein denaturation and aggregation. To combat these effects and ensure survival, cells possess an array of molecular chaperones and proteases. Clp/Hsp100 proteins are one of the major classes of chaperones belonging to the AAAϩ superfamily (ATPases associated with various cellular activities) (1, 2). Two members of this family, Escherichia coli ClpB and its eukaryotic homolog yeast Hsp104, are essential for cell survival during extreme heat stress (3, 4) where they function to disaggregate and reactivate aggregated proteins (5, 6). In vitro ClpB and Hsp104 are able to solubilize and reactivate protein aggregates in ATP-dependent reactions in collaboration with the DnaK/Hsp70 chaperone system, comprising DnaK, DnaJ, and GrpE in prokaryotes and Hsp70, Hsp40, and GrpE-like proteins in eukaryotes (7-10). However, the roles of the two chaperone systems in disaggregation are not well understood.ClpB and Hsp104 are hexameric proteins consisting of identical protomers, with each protomer containing an N-domain and two AAAϩ domains [nucleotide-binding domain 1 and 2 (NBD-1 and NBD-2)] (Fig. 1A and ref. 11). The protomers are arranged in a ring with an axial pore (channel) similar to that observed for other Hsp100 proteins, including ClpA, ClpX, and HslU (11-15). ATP binding, which occurs at the interface of adjacent subunits, stabilizes the hexamer (11, 16). ATP binding is also required for substrate interaction with conserved residues in the pore of hexameric ClpB (17, 18). ClpB and Hsp104 possess a long coiled-coil middle domain inserted in the linear amino acid sequence of NBD-1. From single molecule reconstructions of electron microscopic images the domain seems t...

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Section: Discussionmentioning

confidence: 65%

“…1B). Experiments have implicated the middle domain in coordinating the communication between NBD-1 and NBD-2 (20) as well as the interaction of ClpB with the DnaK system (11,21).…”

mentioning

confidence: 99%

Collaboration between the ClpB AAA+ remodeling protein and the DnaK chaperone system

Doyle

Hoskins

Wickner³

2007

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

104

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“…Although the physical nature of this interaction remains unknown, an ATP-driven M-domain motion has been demonstrated previously for ClpB (35,47). It is tempting to speculate that Hsp70 binding to the M-domain stabilizes or alters an M-domain conformation from a normally inactive to an active state.…”

Section: Discussionmentioning

confidence: 78%

“…The Hsp104 M-domain coiled-coil is composed of four α-helices, which can be subdivided into two smaller coiled-coils, termed motif 1 and motif 2 (5,6). It was previously shown that mutations in the ClpB M-domain helix 2 had a small but significant effect on substrate recovery (34), whereas mutations in helix 3 abrogated protein disaggregation (35), which was attributed to a potentially impaired interaction between ClpB and DnaK (35).…”

Section: Resultsmentioning

confidence: 99%

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.

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104

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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“…Catalytic Turnover Triggers Subunit Exchange-Dissociation of the proteins making up the ring structure has been suggested as a possible mechanism of AAAϩ proteins releasing their product after catalysis (28). Magnesium chelatase is unusual in the sense that Mg-protoporphyrin IX rather than a protein is the ultimate product of the AAAϩ activity, although the BchH subunit is the target of the BchID complex.…”

Section: Mutant-derived Bchi and Wild-type Bchd Form A Complex-r Capmentioning

confidence: 99%

Catalytic Turnover Triggers Exchange of Subunits of the Magnesium Chelatase AAA+ Motor Unit

Lundqvist

Braumann

Kurowska

et al. 2013

Journal of Biological Chemistry

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M Domains Couple the ClpB Threading Motor with the DnaK Chaperone Activity

Cited by 156 publications

References 34 publications

Collaboration between the ClpB AAA+ remodeling protein and the DnaK chaperone system

Collaboration between the ClpB AAA+ remodeling protein and the DnaK chaperone system

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

Catalytic Turnover Triggers Exchange of Subunits of the Magnesium Chelatase AAA+ Motor Unit

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