Unique Peptide Substrate Binding Properties of 110-kDa Heat-shock Protein (Hsp110) Determine Its Distinct Chaperone Activity

Xu, Xinping; Sarbeng, Evans Boateng; Vorvis, Christina; Kumar, Divya P.; Liu, Zhou; Liu, Qinglian

doi:10.1074/jbc.m111.275057

Cited by 92 publications

(133 citation statements)

References 66 publications

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“…The intrinsic peptide substratebinding activity is represented by the peptide substratebinding affinity in the presence of ADP. Consistent with published results (Kumar et al 2011;Xu et al 2012), the binding affinity for this F-NR peptide for WT DnaK was around 1.69 μM in the presence of ADP (Fig. 3b, c).…”

Section: Resultssupporting

confidence: 91%

A disulfide-bonded DnaK dimer is maintained in an ATP-bound state

Liu

Yang

et al. 2017

Cell Stress and Chaperones

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DnaK, a major Hsp70 molecular chaperones in Escherichia coli, is a widely used model for studying Hsp70s. We recently solved a crystal structure of DnaK in complex with ATP and showed that DnaK was packed as a dimer in the crystal structure. Our previous biochemical studies supported the formation of a specific DnaK dimer as observed in the crystal structure in solution in the presence of ATP and suggested an important role of this dimer in efficient interaction with Hsp40 co-chaperones. In this study, we dissected the biochemical properties of this DnaK dimer. To restrict DnaK in this dimer form, we mutated two residues on the dimer interface to cysteine, A303C, and H541C. Upon oxidation, this DnaK-A303C-H541C protein formed a specific dimer linked by disulfide bonds formed between A303C and H541C only in the presence of ATP, consistent with the crystal structure. Intriguingly, this disulfide-bond-linked dimer of DnaK-A303C-H541C has reduced ATPase activity and decreased affinity for peptide substrate. More interestingly, unlike wild-type DnaK, the peptide substrate-binding kinetics of this dimer is drastically accelerated even in the absence of ATP, suggesting this dimer is restricted in an ATP-bound conformation regardless of nucleotide bound, which was further supported by our analysis using tryptophan fluorescence and ATP-induced peptide release. Thus, formation of the dimer restricted DnaK in an ATP-bound state and blocked the progression through the chaperone cycle. Productive progression through the chaperone cycle requires the dissociation of this transient dimer. Surprisingly, a significantly compromised interaction with Hsp40 co-chaperone was observed for this disulfide-bond-linked dimer. Thus, dissociation of this DnaK dimer is equally crucial for efficient Hsp40 interaction. An initial interaction between Hsp70 and Hsp40 requires the formation of DnaK dimer; but a stable Hsp70-Hsp40 interaction may follow the dissociation of the dimer.

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Section: Resultssupporting

confidence: 91%

A disulfide-bonded DnaK dimer is maintained in an ATP-bound state

Liu

Yang

et al. 2017

Cell Stress and Chaperones

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“…An allosteric communication between the NBD and the substrate-binding domain of Hsp110 has been recently reported (54). Our dose responses further showed that, as suggested from the crystal structure of yeast Hsp110 with bovine Hcp70 NBD, equimolar Hsp70 and Hsp110 can optimally refold stable aggregated luciferase, likely by binding to orthologous topological sites in their respective NBDs and by effective reciprocal allosteric signals of similar quality and intensities between the two orthologous SBDs (42).…”

Section: Discussionsupporting

confidence: 75%

“…Like Hsp70s, Hsp110 can hydrolyze ATP (33) and is activated by J-domain co-chaperones (34). Hsp110 can also bind nascent polypeptide chains (33,34,53) and small peptides with different specificities (54). By doing so, Hsp110 may act as a "holding" chaperone that passively prevents the aggregation of stress-or mutation-induced misfolding and aggregating proteins (55).…”

Section: Discussionmentioning

confidence: 99%

Hsp110 Is a Bona Fide Chaperone Using ATP to Unfold Stable Misfolded Polypeptides and Reciprocally Collaborate with Hsp70 to Solubilize Protein Aggregates

Mattoo

Sharma

Priya

et al. 2013

Journal of Biological Chemistry

144

168

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Background: Hsp110s are considered as mere nucleotide exchange factors of the Hsp70s. Results: Human cytosolic Hsp110s can use ATP to unfold misfolded polypeptides and act as equal partner with Hsp70 to solubilize stable protein aggregates. Conclusion: Hsp110s are Hsp70-like polypeptide unfolding chaperones. Significance: Hsp110s are powerful disaggregating chaperones that can collaborate with Hsp70s to detoxify misfolding proteins in degenerative diseases.

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“…To monitor long-range changes upon perturbation of L 34 , we performed CSP analysis on a previously characterized DnaK construct deficient for peptide binding (9). For this construct, called DnaK-L 34 , the entire L 34 (TAEDNQS) was replaced by a shorter sequence (MGG) from a DnaK homologous protein, Hsp110 (9,27). In the domain-undocked conformation, this construct has low substrate affinity-similar to domain-docked (ATP-bound) WT DnaK (27).…”

Section: Resultsmentioning

confidence: 99%

Substrate-binding domain conformational dynamics mediate Hsp70 allostery

Zhuravleva

Gierasch

2015

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

105

164

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Binding of ATP to the N-terminal nucleotide-binding domain (NBD) of heat shock protein 70 (Hsp70) molecular chaperones reduces the affinity of their C-terminal substrate-binding domain (SBD) for unfolded protein substrates. ATP binding to the NBD leads to docking between NBD and βSBD and releasing of the α-helical lid that covers the substrate-binding cleft in the SBD. However, these structural changes alone do not fully account for the allosteric mechanism of modulation of substrate affinity and binding kinetics. Through a multipronged study of the Escherichia coli Hsp70 DnaK, we found that changes in conformational dynamics within the βSBD play a central role in interdomain allosteric communication in the Hsp70 DnaK. ATP-mediated NBD conformational changes favor formation of NBD contacts with lynchpin sites on the βSBD and force disengagement of SBD strand β8 from strand β7, which leads to repacking of a βSBD hydrophobic cluster and disruption of the hydrophobic arch over the substrate-binding cleft. In turn, these structural rearrangements drastically enhance conformational dynamics throughout the entire βSBD and particularly around the substrate-binding site. This negative, entropically driven allostery between two functional sites of the βSBD-the NBD binding interface and the substrate-binding site-confers upon the SBD the plasticity needed to bind to a wide range of chaperone clients without compromising precise control of thermodynamics and kinetics of chaperone-client interactions. molecular chaperone | protein quality control | NMR chemical shift perturbations | conformational selection | entropically driven allostery H eat shock protein 70 (Hsp70) molecular chaperones are central players in protein quality control systems for organisms from bacteria to humans (1, 2). All Hsp70s consist of two highly conserved domains: an N-terminal nucleotide-binding domain (NBD), which regulates the affinity of substrate binding, and a C-terminal substrate-binding domain (SBD), which binds to exposed hydrophobic stretches of client proteins and is made up of a β-sandwich domain (the βSBD), and an α-helical lid (the αLid) (Fig. 1A). Hsp70 functions rely on interdomain allostery: ATP hydrolysis in the NBD controls thermodynamics and kinetics of substrate binding and release; in turn, substrate binding to the SBD stimulates ATPase activity in the NBD (3-5).Mechanistic understanding of Hsp70 function has been greatly enhanced by recent breakthrough achievements in structural characterization of individual functional steps of the allosteric cycle for the Escherichia coli Hsp70 DnaK. New crystal structures and NMR analysis of DnaK have provided descriptions of three major functional states: ADP-bound (6, 7), 9), and ATP/substrate-bound (10), which have distinct arrangements of the four structural units NBD, βSBD, αLid, and interdomain linker (Fig. 1A). In the ADP-bound (domain-undocked, linker-unbound) state, the two DnaK domains, the NBD and SBD, behave independently, with the interdomain linker exposed to the solvent and ...

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Unique Peptide Substrate Binding Properties of 110-kDa Heat-shock Protein (Hsp110) Determine Its Distinct Chaperone Activity

Cited by 92 publications

References 66 publications

A disulfide-bonded DnaK dimer is maintained in an ATP-bound state

A disulfide-bonded DnaK dimer is maintained in an ATP-bound state

Hsp110 Is a Bona Fide Chaperone Using ATP to Unfold Stable Misfolded Polypeptides and Reciprocally Collaborate with Hsp70 to Solubilize Protein Aggregates

Substrate-binding domain conformational dynamics mediate Hsp70 allostery

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