Crystal Structures of the ATPase Domains of Four Human Hsp70 Isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78

Wiśniewska, M.; Karlberg, T.; Lehtiö, L.; Johansson, Ida; Kotenyova, T.; Moche, M.; Schüler, H.

doi:10.1371/journal.pone.0008625

Cited by 132 publications

(106 citation statements)

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“…On the one hand, these results confirm the universal applicability of these degenerate oligonucleotides (see also Schill et al 2004;Reuner et al 2008). Particularly, the targeted, highly conserved consensus region codes for the N-terminal nucleotide-binding domain of the hsp70 proteins (Conserved Domain Database, http://www.ncbi.nlm.nih.gov/Structure/ cdd/cdd.shtml; (Marcheler-Bauer et al 2011), which regulates chaperone activity by ATP hydrolysis (Wisniewska et al 2010). This domain is considered to be less variable than the also well-conserved peptide-binding domain (Reddy et al 2010).…”

Section: Resultssupporting

confidence: 62%

An intron-containing, heat-inducible stress-70 gene in the millipede Tachypodoiulus niger (Julidae, Diplopoda)

Knigge

Bachmann

Köhler

2014

Cell Stress and Chaperones

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The highly conserved part of the nucleotidebinding domain of the hsp70 gene family was amplified from the soil diplopod Tachypodoiulus niger (Julidae, Diplopoda). Genomic DNA yielded 701, 549 and 540 bp sequences, whereas cDNA from heat shocked animals produced only one distinct fragment of 543 bp. The sequences could be classified as a 70 kDa heat shock protein (hsp70), the corresponding 70 kDa heat shock cognate (hsc70) and a glucoserelated hsp70 homologue (grp78). Comparisons of genomic and cDNA sequences of hsc70 identified two introns within the consensus sequence. Generally, stress-70 expression levels were low, which hampered successful RT-PCR and subsequent subcloning. Following experimental heat shock, however, the spliced hsc70 was amplified predominantly, instead of its inducible homologue hsp70. This finding suggests that microevolution in this soil-dwelling arthropod is directed towards low constitutive stress-70 levels and that the capacity for stress-70 induction presumably is limited. hsc70, albeit having introns, apparently is inducible and contributes to the stress-70 response.

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Section: Resultssupporting

confidence: 62%

An intron-containing, heat-inducible stress-70 gene in the millipede Tachypodoiulus niger (Julidae, Diplopoda)

Knigge

Bachmann

Köhler

2014

Cell Stress and Chaperones

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“…The purification of HtpG and Hsp82, Hsp90␣, Grp94, BiP, and aha1 has been described previously (6,13,19). Briefly, Hsp90 proteins were expressed in Escherichia coli strain BL21* at 37°C in LB culture medium and induced with 1 mM isopropyl ␤-D-1-thiogalactopyranoside.…”

Section: Methodsmentioning

confidence: 99%

Crowding Activates Heat Shock Protein 90

Halpin

Huang

Sun

et al. 2016

Journal of Biological Chemistry

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Hsp90 is a dimeric ATP-dependent chaperone involved in the folding, maturation, and activation of diverse target proteins. Extensive in vitro structural analysis has led to a working model of Hsp90's ATP-driven conformational cycle. An implicit assumption is that dilute experimental conditions do not significantly perturb Hsp90 structure and function. However, Hsp90 undergoes a dramatic open/closed conformational change, which raises the possibility that this assumption may not be valid for this chaperone. Indeed, here we show that the ATPase activity of Hsp90 is highly sensitive to molecular crowding, whereas the ATPase activities of Hsp60 and Hsp70 chaperones are insensitive to crowding conditions. Polymer crowders activate Hsp90 in a non-saturable manner, with increasing efficacy at increasing concentration. Crowders exhibit a non-linear relationship between their radius of gyration and the extent to which they activate Hsp90. This experimental relationship can be qualitatively recapitulated with simple structure-based volume calculations comparing open/closed configurations of Hsp90. Thermodynamic analysis indicates that crowding activation of Hsp90 is entropically driven, which is consistent with a model in which excluded volume provides a driving force that favors the closed active state of Hsp90. Multiple Hsp90 homologs are activated by crowders, with the endoplasmic reticulumspecific Hsp90, Grp94, exhibiting the highest sensitivity. Finally, we find that crowding activation works by a different mechanism than co-chaperone activation and that these mechanisms are independent. We hypothesize that Hsp90 has a higher intrinsic activity in the cell than in vitro.Molecular chaperones play a central role in maintaining folded and active proteins in the cell. Hsp70 class chaperones inhibit misfolding by binding and releasing short hydrophobic segments of unstructured polypeptides with cycles of ATP hydrolysis. Hsp60 class chaperonins promote folding by isolating single protein chains within an isolated cavity. Similar to Hsp70 and Hsp60, Hsp90 has an essential ATPase activity, but the underlying functional mechanism appears to be different. Hsp90 plays important regulatory roles under non-stress conditions by its interactions with specific classes of substrates ("clients") such as kinases and nuclear receptors (1). Hsp90 has a dimeric structure that can undergo dramatic rearrangements upon ATP binding and hydrolysis. Despite significant progress in characterizing Hsp90's ATP-dependent conformational cycle in vitro, it is still unclear how Hsp90 performs its many critical cellular functions.Hsp90 conformational heterogeneity results from rigid-body rearrangements of the three domains within the monomer. The N-terminal domain (site of nucleotide binding) can rotate relative to the middle domain. The middle domain can be positioned against the C-terminal domain (site of dimerization) in multiple orientations, resulting in a flexible and structurally heterogeneous open conformation. Indeed, all full-length Hsp90 str...

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“…These structures have shown the structural basis of each domain in binding its substrates. The NBD is made of two lobes, between which is the nucleotide-binding pocket (Flaherty et al 1990;Harrison et al 1997;Sriram et al 1997;Jiang et al 2007;Wisniewska et al 2010). SBD is further divided into two subdomains: SBDβ and SBDα Cupp-Vickery et al 2004;Zhang et al 2014;Clerico et al 2015).…”

Section: Introductionmentioning

confidence: 99%

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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Crystal Structures of the ATPase Domains of Four Human Hsp70 Isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78

Cited by 132 publications

References 50 publications

An intron-containing, heat-inducible stress-70 gene in the millipede Tachypodoiulus niger (Julidae, Diplopoda)

An intron-containing, heat-inducible stress-70 gene in the millipede Tachypodoiulus niger (Julidae, Diplopoda)

Crowding Activates Heat Shock Protein 90

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

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