Motor Mechanism for Protein Threading through Hsp104

Wendler, Petra; Shorter, James; Snead, David; Plisson, Célia; Clare, Daniel K.; Lindquist, Susan; Saibil, Helen R.

doi:10.1016/j.molcel.2009.02.026

Cited by 85 publications

(164 citation statements)

References 45 publications

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“…3G), provide additional support for this model. In this regard, we note that there is also strong evidence that the doublering ClpB and Hsp-104 disaggregation chaperones operate by using translocation mechanisms (25,26). Although these results do not rule out alternative models, we believe that many mechanical remodeling functions of Cdc48/p97 can be explained by a polypeptide-translocation mechanism.…”

Section: Resultsmentioning

confidence: 80%

Architecture and assembly of the archaeal Cdc48⋅20S proteasome

Barthelme¹,

Chen²,

Grabenstatter

et al. 2014

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

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ATP-dependent proteases maintain protein quality control and regulate diverse intracellular functions. Proteasomes are primarily responsible for these tasks in the archaeal and eukaryotic domains of life. Even the simplest of these proteases function as large complexes, consisting of the 20S peptidase, a barrel-like structure composed of four heptameric rings, and one or two AAA+ (ATPase associated with a variety of cellular activities) ring hexamers, which use cycles of ATP binding and hydrolysis to unfold and translocate substrates into the 20S proteolytic chamber. Understanding how the AAA+ and 20S components of these enzymes interact and collaborate to execute protein degradation is important, but the highly dynamic nature of prokaryotic proteasomes has hampered structural characterization. Here, we use electron microscopy to determine the architecture of an archaeal Cdc48·20S proteasome, which we stabilized by site-specific cross-linking. This complex displays coaxial alignment of Cdc48 and 20S and is enzymatically active, demonstrating that AAA+ unfoldase wobbling with respect to 20S is not required for function. In the complex, the N-terminal domain of Cdc48, which regulates ATP hydrolysis and degradation, packs against the D1 ring of Cdc48 in a coplanar fashion, constraining mechanisms by which the N-terminal domain alters 20S affinity and degradation activity.AAA+ protease | dynamic wobbling model | p97/VCP P roteasomes are large macromolecular complexes that degrade misfolded or damaged proteins to maintain cellular homeostasis and quality control in all domains of life. In addition, selective proteasomal turnover of regulatory proteins is often a critical element of signaling cascades that allow cells to respond to changing conditions and environmental stress (1, 2). Proteasomes consist of the self-compartmentalized 20S peptidase and one or two AAA+ (ATPase associated with a variety of cellular activities) family ring hexamers. The α 7 β 7 β 7 α 7 ring topology of the 20S enzyme ensures that the proteolytic active sites, which reside in the β-subunits, are sequestered and can only cleave substrates that enter the chamber through narrow axial pores formed by the α-subunits (3). As a consequence, ATP-dependent unfolding of natively folded protein substrates by the AAA+ ring and subsequent polypeptide translocation through a narrow axial channel and into the 20S chamber are required for degradation (4).Although the 20S peptidase is the degradation module in all proteasomes, the associated AAA+ unfolding machines differ. For example, homohexamers of Mpa/Arc in actinobacteria and PAN in archaea serve as proteasomal motors, whereas a heterohexameric Rpt 1-6 ring in the 19S particle is the motor of the eukaryotic 26S proteasome (2, 5). Characteristic of type-I AAA+ enzymes, Mpa/Arc, PAN, and Rpt 1-6 subunits contain a single AAA+ module for ATP binding and hydrolysis (6). By contrast, as found in type-II AAA+ enzymes, the homohexameric Cdc48 motor in the recently discovered archaeal Cdc48·20S proteasome cont...

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Section: Resultsmentioning

confidence: 80%

Architecture and assembly of the archaeal Cdc48⋅20S proteasome

Barthelme¹,

Chen²,

Grabenstatter

et al. 2014

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

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“…Probabilistic, semisequential, and sequential modes of action have been proposed for other AAAϩ proteins (10,26,27). For example, another hexameric Clp protein, ClpX, has been shown to function in a probabilistic manner by Martin, Baker, and Sauer (26).…”

Section: Discussionmentioning

confidence: 99%

“…For example, another hexameric Clp protein, ClpX, has been shown to function in a probabilistic manner by Martin, Baker, and Sauer (26). In contrast, Saibil, Lindquist, and colleagues recently proposed that Hsp104, the yeast homolog of ClpB, uses a sequential mechanism, based on electron microscopic data showing asymmetry in the hexameric model (10). Crystal structures showing asymmetric conformations have been observed for several AAAϩ helicases, suggesting that those proteins act by a sequential mechanism (27).…”

Section: Discussionmentioning

confidence: 99%

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

Hoskins

Doyle

Wickner

2009

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

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ClpB and Hsp104 are members of the AAA؉ (ATPases associated with various cellular activities) family of proteins and are molecular machines involved in thermotolerance. They are hexameric proteins containing 12 ATP binding sites with two sites per protomer. ClpB and Hsp104 possess some innate protein remodeling activities; however, they require the collaboration of the DnaK/Hsp70 chaperone system to disaggregate and reactivate insoluble aggregated proteins. We investigated the mechanism by which ClpB couples ATP utilization to protein remodeling with and without the DnaK system. When wild-type ClpB, which is unable to remodel proteins alone in the presence of ATP, was mixed with a ClpB mutant that is unable to hydrolyze ATP, the heterohexamers surprisingly gained protein remodeling activity. Optimal protein remodeling by the heterohexamers in the absence of the DnaK system required approximately three active and three inactive protomers. In addition, the location of the active and inactive ATP binding sites in the hexamer was not important. The results suggest that in the absence of the DnaK system, ClpB acts by a probabilistic mechanism. However, when we measured protein disaggregation by ClpB heterohexamers in conjunction with the DnaK system, incorporation of a single inactive ClpB subunit blocked activity, supporting a sequential mechanism of ATP utilization. Taken together, the results suggest that the mechanism of ATP utilization by ClpB is adaptable and can vary depending on the specific substrate and the presence of the DnaK system.B acterial ClpB and yeast Hsp104 are ATP-dependent protein remodeling machines that function to disaggregate protein aggregates and reactivate proteins after extreme stress conditions (1-3). In the cell, ClpB acts in conjunction with the DnaK chaperone system and Hsp104 acts with the Hsp70 chaperone system (4, 5). DnaK and Hsp70 are members of another large, ubiquitous family of ATP-dependent molecular chaperones that mediate protein reactivation and remodeling in concert with two cochaperones, DnaJ and GrpE in prokaryotes and Hsp40 and NEF in eukaryotes (6). Alone, neither the DnaK/Hsp70 chaperone system nor ClpB/Hsp104 has the ability to reactivate large insoluble aggregates.ClpB/Hsp104 exists as a hexameric ring with an axial channel (7-10). Each protomer contains two AAAϩ (ATPases associated with various cellular activities) nucleotide-binding domains separated by a hinge region and preceded by an N-terminal domain (1, 7). The two AAAϩ domains contain characteristic motifs, including Walker A and B and sensor-1 and -2 motifs, as well as an arginine finger (11,12). Situated in the first AAAϩ domain is a long coiled-coil region, referred to as the middle domain, which is unique to ClpB, Hsp104, and their homologs.In vitro ClpB/Hsp104 solubilizes and reactivates protein aggregates in ATP-dependent reactions in collaboration with the DnaK/Hsp70 chaperone system (1-3). Although the roles of the two chaperone systems in disaggregation are not fully understood, it is likely that ...

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“…Steady progress continues to be made on the structure-function relationships and reaction cycles of chaperones bound to individual client proteins [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. In this issue, however, we return to first principles, and shine the spotlight on the role of chaperones in biomolecular assemblies, as first defined for nucleosomes by Laskey [17].…”

Section: Chaperones At the Crossroads Of Life And Deathmentioning

confidence: 99%

Chaperones: needed for both the good times and the bad times

Quinlan

Ellis

2013

Phil. Trans. R. Soc. B

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In this issue, we explore the assembly roles of protein chaperones, mainly through the portal of their associated human diseases (e.g. cardiomyopathy, cataract, neurodegeneration, cancer and neuropathy). There is a diversity to chaperone function that goes beyond the current emphasis in the scientific literature on their undoubted roles in protein folding and refolding. The focus on chaperone-mediated protein folding needs to be broadened by the original Laskey discovery that a chaperone assists the assembly of an oligomeric structure, the nucleosome, and the subsequent suggestion by Ellis that other chaperones may function in assembly processes, as well as in folding. There have been a number of recent discoveries that extend this relatively neglected aspect of chaperone biology to include proteostasis, maintenance of the cellular redox potential, genome stability, transcriptional regulation and cytoskeletal dynamics. So central are these processes that we propose that chaperones stand at the crossroads of life and death because they mediate essential functions, not only during the bad times, but also in the good times. We suggest that chaperones facilitate the success of a species, and hence the evolution of individuals within populations, because of their contributions to so many key cellular processes, of which protein folding is only one.

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Motor Mechanism for Protein Threading through Hsp104

Cited by 85 publications

References 45 publications

Architecture and assembly of the archaeal Cdc48⋅20S proteasome

Architecture and assembly of the archaeal Cdc48⋅20S proteasome

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

Chaperones: needed for both the good times and the bad times

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