The J-Domain of Hsp40 Couples ATP Hydrolysis to Substrate Capture in Hsp70

Wittung-Stafshede, Pernilla; Guidry, Jessie; Horne, B. Erin; Landry, Samuel J.

doi:10.1021/bi027333o

Cited by 102 publications

(113 citation statements)

References 46 publications

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“…3B). This observation is similar to published binding measurements of E. coli DnaK and a J-domain HPD mutant (50). We found that the HPD point mutant can prevent J protein from fully activating DnaK when aggregates are absent (SI Appendix, Fig.…”

Section: Discussionsupporting

confidence: 90%

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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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Section: Discussionsupporting

confidence: 90%

“…3B, Lower). Studies in yeast and E. coli have also demonstrated that HPD J-protein point mutants interact with DnaK homologs (46,50).…”

Section: Resultsmentioning

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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“…However, this difference in findings is likely to be due to the fact that there was no ATP present in assay conditions in those previous studies, whereas the binding of the HSPs was examined here in the presence of physiological levels of ATP. HSP72 is an ATPase that in its ATP-bound state has a low affinity and high exchange rate with target proteins, but such interaction catalyzes the hydrolysis of the ATP to ADP, resulting in a conformational change in the HSP72 that greatly slows its dissociation from the target until the ADP is displaced by ATP (22,23,48). Thus, in the present study, the heat treatment of the muscles possibly produced S-glutathionylation of most of the HSP72, leading to it tightly associating with target proteins within the fibers (probably predominantly SERCA), and this could be subsequently reversed by application of strongly reducing conditions (10 mM DTT) in the presence of ATP.…”

Section: Discussionmentioning

confidence: 99%

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers

Larkins

Murphy

Lamb

2012

American Journal of Physiology-Cell Physiology

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Larkins NT, Murphy RM, Lamb GD. Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers. Am J Physiol Cell Physiol 303: C654 -C665, 2012. First published July 3, 2012; doi:10.1152/ajpcell.00180.2012.-Heat shock proteins (HSPs) help maintain cellular function in stressful situations, but the processes controlling their interactions with target proteins are not well defined. This study examined the binding of HSP72, HSP25, and ␣B-crystallin in skeletal muscle fibers following various stresses. Rat soleus (SOL) and extensor digitorum longus (EDL) muscles were subjected in vitro to heat stress or strongly fatiguing stimulation. Superficial fibers were "skinned" by microdissection and HSP diffusibility assessed from the extent of washout following 10-to 30 min exposure to a physiological intracellular solution. In fibers from nonstressed (control) SOL muscle, Ͼ80% of each HSP is readily diffusible. However, after heating a muscle to 40°C for 30 min ϳ95% of HSP25 and ␣B-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to Ն44°C. The ratio of reduced to oxidized cytoplasmic glutathione (GSH:GSSG) decreased approximately two-and fourfold after heating muscles to 40°and 45°C, respectively. The reducing agent dithiothreitol reversed HSP72 binding in heated muscles but had no effect on the other HSPs. Intense in vitro stimulation of SOL muscles, sufficient to elicit substantial oxidation-related loss of maximum force and approximately fourfold decrease in the GSH:GSSG ratio, had no effect on diffusibility of any of the HSPs. When skinned fibers from heat-treated muscles were bathed with additional exogenous HSP72, total binding increased approximately two-and 10-fold, respectively, in SOL and EDL fibers, possibly reflective of the relative sarco(endo)plasmic reticulum Ca 2ϩ -ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on ␣B-crystallin and Ser85 on HSP25 increased with heat treatment but did not appear to determine HSP binding. The findings highlight major differences in the processes controlling binding of HSP72 and the two small HSPs. Binding was not directly related to cytoplasmic oxidative status, but oxidation of cysteine residues influenced HSP72 binding. oxidation; stress response; skinned fiber; chaperones HEAT SHOCK PROTEINS (HSPs) are highly conserved and ubiquitously expressed proteins that act as protein chaperones and in stress situations bind and interact with various proteins to help preserve or restore their function (23,32). A great deal of work in skeletal muscle has focused on the increase in HSP protein and/or mRNA expression levels occurring in hours to days after various stressful stimuli, such as eccentric exercise (11,17,26,27,39), oxidative stress (50), heat (4, 35), and glycogen depletion (12). The present study focuses instead on the acute responses of HSPs to stresses, examining the effects of heat, contraction, and o...

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“…The two functions of DnaJ have been assigned to the C terminus and N terminus of the molecular chaperone, respectively. The N-terminal 75-residue J-domain (Jd) 2 is responsible for stimulation of DnaK ATPase activity (14,15). The Jd is also able to couple ATP hydrolysis to peptide capture in DnaK (15).…”

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confidence: 99%

The Hsp40 J-domain Stimulates Hsp70 When Tethered by the Client to the ATPase Domain

Horne

Genevaux³

et al. 2010

Journal of Biological Chemistry

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Hsp70-family molecular chaperones have numerous constitutive and stress-induced roles that involve binding to short hydrophobic stretches within a client protein. Protein folding, protein translocation, and protein disaggregation are a few of the functions that Hsp70s carry out under normal conditions within the cell (1-4).The two major activities of Hsp70s are located in the two major domains of the protein. The N-terminal 44-kDa domain binds and hydrolyzes ATP, and the 23-kDa peptide binding domain binds client proteins (5). The peptide binding domain is divided into the ␤-sandwich subdomain where client binding occurs and the ␣-helical subdomain, which acts as a lid on the ␤-sandwich subdomain (6). The two major domains are connected by a highly conserved linker region.Escherichia coli Hsp70/DnaK is the most extensively studied member of the Hsp70s. The behavior of DnaK is like other Hsp70s in that its binding to client proteins is regulated by binding and hydrolysis of ATP. The binding of ATP converts DnaK to the low affinity conformation. This form of the protein binds and releases client proteins quickly, on the time scale of seconds (7,8). Hydrolysis of ATP slows the off-rate of client proteins by 3 orders of magnitude compared with when ATP is bound (9). Previous studies have shown that a portion of the lid subdomain of DnaK modulates the allosteric behavior. For example, for a lidless form of DnaK (DnaK(1-517)) containing only a portion of helix A, the ATP-stimulated release of peptide is ϳ40-fold faster than for wild-type DnaK (10).The DnaK client binding cycle is regulated by a nucleotide exchange factor, GrpE (11). GrpE interacts with the ATPase domain of DnaK to catalyze the release of ADP (12). Upon release of ADP, DnaK binds ATP, which stimulates the release of bound client.The structurally and functionally distinct E. coli Hsp40/DnaJ has been described as a co-chaperone for Hsp70/DnaK, and the two proteins are said to operate as a single chaperone machine (13). DnaJ binds to client proteins, and it stimulates the DnaK ATPase activity. The two functions of DnaJ have been assigned to the C terminus and N terminus of the molecular chaperone, respectively. The N-terminal 75-residue J-domain (Jd) 2 is responsible for stimulation of DnaK ATPase activity (14, 15). The Jd is also able to couple ATP hydrolysis to peptide capture in DnaK (15). Two distinct models have been suggested for how the Jd couples the DnaK ATPase and peptide binding activities, client protein-recruitment, and triggered capture. In the recruitment model, the Jd brings an associated client protein to . In the triggered-capture model, the client protein brings the Jd to DnaK (14,19,20). After formation of the three-component complex, the Jd stimulates ATP hydrolysis by DnaK, which firmly stabilizes the client-bound conformation of DnaK. The triggered-capture model more accurately described the role of the Jd in binding of a model client, the peptide p5, because the association rate of the p5 with DnaK was only modestly increased by ...

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The J-Domain of Hsp40 Couples ATP Hydrolysis to Substrate Capture in Hsp70

Cited by 102 publications

References 46 publications

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

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

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers

The Hsp40 J-domain Stimulates Hsp70 When Tethered by the Client to the ATPase Domain

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