Action of the Hsp70 chaperone system observed with single proteins

Nunes, João M.; Mayer-Hartl, Manajit; Hartl, F. Ulrich; Müller, Daniel J.

doi:10.1038/ncomms7307

Cited by 63 publications

(58 citation statements)

References 47 publications

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“…We observe that 11% of refolding attempts yield non-I91 unfolding events. Previous literature has detected similar types of events, which were determined to be misfolded I91 (82)(83)(84)(85).…”

Section: Luciferase Initiates Refolding Through Folding Nucleimentioning

confidence: 71%

Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase

Scholl

Yang

Marszałek

2017

Biophysical Journal

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Proteins obtain their final functional configuration through incremental folding with many intermediate steps in the folding pathway. If known, these intermediate steps could be valuable new targets for designing therapeutics and the sequence of events could elucidate the mechanism of refolding. However, determining these intermediate steps is hardly an easy feat, and has been elusive for most proteins, especially large, multidomain proteins. Here, we effectively map part of the folding pathway for the model large multidomain protein, Luciferase, by combining single-molecule force-spectroscopy experiments and coarsegrained simulation. Single-molecule refolding experiments reveal the initial nucleation of folding while simulations corroborate these stable core structures of Luciferase, and indicate the relative propensities for each to propagate to the final folded native state. Both experimental refolding and Monte Carlo simulations of Markov state models generated from simulation reveal that Luciferase most often folds along a pathway originating from the nucleation of the N-terminal domain, and that this pathway is the least likely to form nonnative structures. We then engineer truncated variants of Luciferase whose sequences corresponded to the putative structure from simulation and we use atomic force spectroscopy to determine their unfolding and stability. These experimental results corroborate the structures predicted from the folding simulation and strongly suggest that they are intermediates along the folding pathway. Taken together, our results suggest that initial Luciferase refolding occurs along a vectorial pathway and also suggest a mechanism that chaperones may exploit to prevent misfolding.

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Section: Luciferase Initiates Refolding Through Folding Nucleimentioning

confidence: 71%

Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase

Scholl

Yang

Marszałek

2017

Biophysical Journal

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“…Switching between low-and high-affinity states for substrate peptides is key for the function of Hsp70 chaperones (9). The closed state binds firmly to peptide clients to prevent aggregation, whereas the open state must release the peptides to allow them to fold (5).…”

Section: Discussion Bifurcating Unfolding Pathways For Sbd and Mechanmentioning

confidence: 99%

“…1A; refs. 7 and 8) to allow exchange of substrates (9). Several crystal structures of the isolated SBD (in which the NBD is absent) have been solved (10)(11)(12)(13)(14)(15).…”

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confidence: 99%

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change

Mandal

Merz

Buchsteiner

et al. 2017

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

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Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α-and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.laser trapping | parallel pathways | elasticity | force | protein extension W hen looking at protein structures at atomic resolution, it is often tempting to use macroscopic mechanical analogies to describe their function as molecular machines. However, such analogies are often misleading because boundaries between independently stable subdomains cannot often be determined from structures, owing to the high cooperativity of protein folding and structural transitions. Single-molecule protein nanomechanics have emerged as a tool to force biomolecules through their conformational space and, hence, identify hinges, breaking points, and mechanically stable subdomains (1-3).A prominent example of a protein machine undergoing large conformational change during its functional cycle is the ATP-regulated Hsp70 chaperone DnaK-a central molecular chaperone of the protein quality control network in a cell (4-6). Once ATP is bound to the nucleotide binding domain (NBD, blue-yellow; Fig. 1A) of DnaK, the initially closed substrate binding domain (SBD) opens its binding cleft by engaging the β-subdomain to the NBD. In doing so, it undergoes a dramatic ∼10 Å displacement of its lid subdomain ( Fig. 1A; refs. 7 and 8) to allow exchange of substrates (9). Several crystal structures of the isolated SBD (in which the NBD is absent) have been solved (10-15). In these structures, the absence or presence of peptide clients or nonnatural ligands induce no significant structural changes in the closed conformation. There is no indication in the crystal structures of the huge conformational change of the lid domain of the SBD, seen in the ATP form of the full-length two-domain DnaK. Therefore, the large conformational change of the SBD is only observed in the two-domain DnaK after ATP binding. Thus, although the crystal structures provide us with valuable insights into the 3D arrangement of individual atoms, the thermodynamic and mechanical stability of individual substructures are difficult to predict based on this information alone. Here, we ask how the large ATP-induced changes of the SBD, as seen in the two-domain DnaK, are mirrored in the subdomain integrity and nano...

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“…The multiple J proteins of eukaryotes are believed to increase the ability of Hsp70 to recognize diverse substrates, albeit with some redundancy (31). Different eukaryotic J proteins produce varying effects on in vitro Hsp70 ATPase and refolding activities (32)(33)(34), or can act as holdases that prevent protein aggregation (35,36). Unique mixtures of J proteins can function as disaggregases (37,38).…”

Section: Significancementioning

confidence: 99%

Reconstitution of a Mycobacterium tuberculosis proteostasis network highlights essential cofactor interactions with chaperone DnaK

Lupoli

Fay

Adura

et al. 2016

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

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During host infection, Mycobacterium tuberculosis (Mtb) encounters several types of stress that impair protein integrity, including reactive oxygen and nitrogen species and chemotherapy. The resulting protein aggregates can be resolved or degraded by molecular machinery conserved from bacteria to eukaryotes. Eukaryotic Hsp104/Hsp70 and their bacterial homologs ClpB/DnaK are ATP-powered chaperones that restore toxic protein aggregates to a native folded state. DnaK is essential in Mycobacterium smegmatis, and ClpB is involved in asymmetrically distributing damaged proteins during cell division as a mechanism of survival in Mtb, commending both proteins as potential drug targets. However, their molecular partners in protein reactivation have not been characterized in mycobacteria. Here, we reconstituted the activities of the Mtb ClpB/DnaK bichaperone system with the cofactors DnaJ1, DnaJ2, and GrpE and the small heat shock protein Hsp20. We found that DnaJ1 and DnaJ2 activate the ATPase activity of DnaK differently. A point mutation in the highly conserved HPD motif of the DnaJ proteins abrogates their ability to activate DnaK, although the DnaJ2 mutant still binds to DnaK. The purified Mtb ClpB/DnaK system reactivated a heat-denatured model substrate, but the DnaJ HPD mutants inhibited the reaction. Finally, either DnaJ1 or DnaJ2 is required for mycobacterial viability, as is the DnaK-activating activity of a DnaJ protein. These studies lay the groundwork for strategies to target essential chaperone–protein interactions in Mtb, the leading cause of death from a bacterial infection.

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Action of the Hsp70 chaperone system observed with single proteins

Cited by 63 publications

References 47 publications

Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase

Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change

Reconstitution of a Mycobacterium tuberculosis proteostasis network highlights essential cofactor interactions with chaperone DnaK

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