Interplay between E. coli DnaK, ClpB and GrpE during Protein Disaggregation

Doyle, Shannon M.; Shastry, Shankar; Kravats, Andrea N.; Shih, Yu-Hsuan; Miot, Marika; Hoskins, Joel R.; Stan, George; Wickner, Sue

doi:10.1016/j.jmb.2014.10.013

Cited by 58 publications

(62 citation statements)

References 64 publications

(126 reference statements)

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“…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).…”

mentioning

confidence: 99%

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

Rosenzweig

Farber

Vėlyvis

et al. 2015

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

107

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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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mentioning

confidence: 99%

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

Rosenzweig

Farber

Vėlyvis

et al. 2015

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

107

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“…This interaction might facilitate substrate transfer between both chaperones, taking advantage of the opposite affinities for substrate proteins of their ADP and ATP states. This does not rule out the possibility that the DnaK NBD could also interact with ClpB, as seen by NMR for the Thermus homologues and by a recent mutagenesis study showing that DnaK variants with substitutions in subdomains IB and IIB were defective in ClpB interaction [69,90]. However, the interaction of ClpB with DnaK NBD did not significantly alter the ATPase activity and dynamics of ClpB, as found for the Hsp70 NBD domain that bound the M domain of Hsp104 without activating the disaggregase [68].…”

Section: Clpb Dynamics and Disaggregase Activitymentioning

confidence: 97%

Chaperone-assisted protein aggregate reactivation: Different solutions for the same problem

Aguado

Fernández-Higuero

Moro

et al. 2015

Archives of Biochemistry and Biophysics

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“…GrpE and ClpB compete for binding to the NBD of DnaK in the absence of crowders [15,16]. Our previous study showed that crowding shifted the association equilibrium of ClpB toward the functional hexamer and favored a compact, ATP-like state of the disaggregase with higher affinity for DnaK, which resulted in an increased reactivation efficiency of the bichaperone complex [17].…”

Section: Crowding Allows Competition Of Clpb With Grpe For Dnak Bindingmentioning

confidence: 98%

“…When the aggregates are formed by extensively denatured proteins in which the unfolded proteins are enriched in intermolecular β-structure, their reactivation requires the concerted action of the DnaK system and the hexameric disaggregase ClpB (Hsp100). The ability of the DnaK system to work independently or associate with ClpB has been proposed to be controlled by the competition between GrpE and ClpB to bind the NBD of DnaK [15,16]. We have recently characterized the effect of macromolecular crowding on the association properties and activity of ClpB and on its interaction with the DnaK system to form the bichaperone complex [17].…”

Section: Introductionmentioning

confidence: 99%