Species-specific collaboration of heat shock proteins (Hsp) 70 and 100 in thermotolerance and protein disaggregation

Miot, Marika; Reidy, Michael; Doyle, Shannon M.; Hoskins, Joel R.; Johnston, Danielle M.; Genest, Olivier; Vitery, Maria del Carmen; Masison, Daniel C.; Wickner, Sue

doi:10.1073/pnas.1102828108

Cited by 134 publications

(182 citation statements)

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“…It is of interest that many of the consensus motifs are enriched in hydrophobic residues (Leu, Ile, Val, Phe, and Tyr), which are often found in Hsp70 substrates (38). Importantly, we found that the location of consensus motifs within M-domain helix 2 (motifs E and F) overlapped with mutation sites in Escherichia coli ClpB, which either impaired ClpB's innate protein disaggregating activity or affected the functional cooperation between ClpB and DnaK/DnaJ/GrpE in substrate recovery (34). For instance, Hsp70 ΔC recognized both Lys442/ Asp443/Arg444 (motif E) and His471/Glu474/Glu475 (motif F) (Fig.…”

Section: Resultsmentioning

confidence: 77%

“…The Hsp104 M-domain coiled-coil is composed of four α-helices, which can be subdivided into two smaller coiled-coils, termed motif 1 and motif 2 (5,6). It was previously shown that mutations in the ClpB M-domain helix 2 had a small but significant effect on substrate recovery (34), whereas mutations in helix 3 abrogated protein disaggregation (35), which was attributed to a potentially impaired interaction between ClpB and DnaK (35).…”

Section: Resultsmentioning

confidence: 99%

“…For instance, Hsp70 ΔC recognized both Lys442/ Asp443/Arg444 (motif E) and His471/Glu474/Glu475 (motif F) (Fig. 1D), which correspond to Lys438/Lys439/Arg440 (Mut-1) and Ala467/Ser470/Gly471 (Mut-4) in ClpB (34). To determine the specificity of the Hsp70-peptide interaction, we replaced Lys442/Asp443/Arg444 with glutamates (mut-1), and His471/ Glu474/Glu475 with alanines (mut-4).…”

Section: Resultsmentioning

confidence: 99%

“…In support of a direct interaction, it was shown that replacing the M-domain of yeast Hsp104 with that of bacterial ClpB is sufficient to switch the species-specificity of the bichaperone system so that the yeast Hsp104 chimera now cooperated with the bacterial Hsp70 system in protein disaggregation, and vice versa (33,34).…”

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confidence: 99%

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Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

Lee¹,

Kim²,

Biter³

et al. 2013

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

102

111

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Heat shock protein (Hsp) 104 is a ring-forming, protein-remodeling machine that harnesses the energy of ATP binding and hydrolysis to drive protein disaggregation. Although Hsp104 is an active ATPase, the recovery of functional protein requires the speciesspecific cooperation of the Hsp70 system. However, like Hsp104, Hsp70 is an active ATPase, which recognizes aggregated and aggregation-prone proteins, making it difficult to differentiate the mechanistic roles of Hsp104 and Hsp70 during protein disaggregation. Mapping the Hsp70-binding sites in yeast Hsp104 using peptide array technology and photo-cross-linking revealed a striking conservation of the primary Hsp70-binding motifs on the Hsp104 middle-domain across species, despite lack of sequence identity. Remarkably, inserting a Strep-Tactin binding motif at the spatially conserved Hsp70-binding site elicits the Hsp104 protein disaggregating activity that now depends on Strep-Tactin but no longer requires Hsp70/40. Consistent with a Strep-Tactin-dependent activation step, we found that full-length Hsp70 on its own could activate the Hsp104 hexamer by promoting intersubunit coordination, suggesting that Hsp70 is an activator of the Hsp104 motor.ClpB | DnaK | Hsp100 | molecular chaperone

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Section: Resultsmentioning

confidence: 77%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

Lee¹,

Kim²,

Biter³

et al. 2013

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

102

111

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“…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 cooperated with the eukaryotic Hsp70/40 system and vice versa (7,27). The role of the M-domain in mediating DnaKJ/GrpE interaction is consistent with the M-domain being on the outside of the ClpB hexamer (5,8), but incompatible with the previously proposed structure of yeast Hsp104 (28,29), which placed the M-domains on the interior or intercalated between subunits.…”

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confidence: 99%

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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Reactivation of Aggregated Proteins by the ClpB/DnaK Bi‐Chaperone System

2016

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Protein aggregation is a common problem in protein biochemistry and is linked to many cellular pathologies and human diseases. The molecular chaperone ClpB can resolubilize and reactivate aggregated proteins. This unit describes the procedure for following reactivation of an aggregated enzyme glucose-6-phosphate dehydrogenase mediated by ClpB from Escherichia coli in cooperation with another molecular chaperone DnaK. The procedures for purification of these chaperones are also described.

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Species-specific collaboration of heat shock proteins (Hsp) 70 and 100 in thermotolerance and protein disaggregation

Cited by 134 publications

References 35 publications

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

Structural basis for intersubunit signaling in a protein disaggregating machine

Reactivation of Aggregated Proteins by the ClpB/DnaK Bi‐Chaperone System

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