DnaK‐mediated association of ClpB to protein aggregates. A bichaperone network at the aggregate surface

Acebrón, Sergio P.; Martín, Ianire; Castillo, Urko del; Moro, Fernando; Muga, Arturo

doi:10.1016/j.febslet.2009.08.020

Cited by 66 publications

(68 citation statements)

References 34 publications

(55 reference statements)

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“…Changes in NTD spectra were not detected upon addition of protein aggregates ( Fig. S1 A and B), in agreement with previous observations that ClpB does not stably bind to aggregated proteins in the absence of the DnaK chaperone system (23,24). Similarly, binding was not observed for globally folded proteins (GFP and DnaK) ( Fig.…”

Section: Resultssupporting

confidence: 79%

ClpB N-terminal domain plays a regulatory role in protein disaggregation

Rosenzweig

Farber

Vėlyvis

et al. 2015

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

114

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ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/ Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD-substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.T he heat shock protein ClpB (Escherichia coli) or Hsp100 (eukaryotes) is the main protein disaggregase in bacteria, yeast, plants, and mitochondria of all eukaryotic cells, and it is essential for cell survival during severe stress (1-4). Recovery of functional proteins from aggregates by ClpB requires the synergistic interaction with a second molecular chaperone, DnaK (1). Through its cochaperone, DnaJ, DnaK initially binds to the aggregates, leading to the exposure of peptide segments that can be recognized by ClpB (5, 6). DnaK then recruits ClpB to the site of aggregation through direct physical interaction (7, 8), transferring the aggregate to ClpB. Using the energy derived from ATP hydrolysis, ClpB unravels the aggregate by threading single polypeptide chains, one at a time, through the central pore of its hexameric ring (9). Once released from the aggregate, the unfolded polypeptides can either refold spontaneously or fold with the help of additional cellular chaperones.Like other Hsp100 proteins, ClpB forms a hexameric ring, with each protomer comprising an N-terminal domain (NTD) and two nucleotide binding domains (NBD1 and NBD2) separated by a unique regulatory coil-coil domain (10) essential for DnaK binding (7, 11) ( Fig. 1 A and B). Both NBDs contain Walk...

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Section: Resultssupporting

confidence: 79%

ClpB N-terminal domain plays a regulatory role in protein disaggregation

Rosenzweig

Farber

Vėlyvis

et al. 2015

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

114

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“…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).…”

mentioning

confidence: 65%

“…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)(22)(23). Once targeted, ClpB disaggregates protein aggregates by extracting unfolded polypeptides (24) and threading them through the ClpB hexamer ring (21,25).…”

mentioning

confidence: 76%

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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“…For example, HtpG can interact with DnaK/DnaJ/GrpE to further promote refolding of aggregated proteins in an ATP-dependent manner [21]. Alternatively, ClpB can also assist in cooperation with DnaK/DnaJ/GrpE in ATP-driven refolding of aggregated proteins [20,22,23]. The small heat shock proteins (sHSPs) IbpA and IbpB stabilize and decrease the size of protein aggregates [24] thereby promoting DnaK/DnaJ/GrpE and ClpB mediated refolding [25] and further possible proteolysis.…”

Section: Cellular Formation Of Ibsmentioning

confidence: 99%

Bacterial Inclusion Bodies: Discovering Their Better Half

Rinas

García‐Fruitós

Corchero

et al. 2017

Trends in Biochemical Sciences

143

123

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Bacterial inclusion bodies are functional, non-toxic amyloids occurring in recombinant bacteria that show analogies with secretory granules of the mammalian endocrine system. The scientific interest in these mesoscale protein aggregates has been historically masked by their status as a hurdle in recombinant protein production.However, insights into their molecular organization and the progressive understanding on how the cell handles the quality of recombinant polypeptides have stimulated the interest on inclusion bodies and opened ways for their usability in diverse technological fields. The engineering and tailoring of inclusion bodies as functional protein particles for materials science and biomedicine is a good example of how formerly undesired bacterial by-products can be re-discovered as promising functional materials for a broad spectrum of applications.-2 - BACKGROUND

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DnaK‐mediated association of ClpB to protein aggregates. A bichaperone network at the aggregate surface

Cited by 66 publications

References 34 publications

ClpB N-terminal domain plays a regulatory role in protein disaggregation

ClpB N-terminal domain plays a regulatory role in protein disaggregation

Structural basis for intersubunit signaling in a protein disaggregating machine

Bacterial Inclusion Bodies: Discovering Their Better Half

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