Insights into the effect of the J-domain on the substrate binding domain (SBD) of the 70 kDa heat shock protein, Hsp70, from a chimeric human J-SBD polypeptide

AbstractDnaJ/Hsp40 chaperones deliver unfolded proteins and stimulate the ATPase activity of DnaK/Hsp70 via their J-domain, a crucial event for the function that this system has in assisting protein folding. The interaction between Hsp40 and Hsp70 is transient and thus difficult to study, since mixing the binding partners can lead to quick dissociation due to their low affinity, creating a challenge for detailed analysis. As a consequence, knowledge of many important aspects of the mechanism of interaction is still lacking, for instance, the effect that J-domain binding has on Hsp70. In this study, we investigated whether it would be possible to gain understanding of this interaction by engineering a chimeric polypeptide where the J-domain of Hsp40 was covalently attached to the substrate binding domain (SBD) of Hsp70 by a flexible linker. The rationale for this is that an increase in the proximity between the interacting partners in this engineered chimera will promote the natural interaction and facilitate the characterization of the protein– protein interaction, which is a requirement to gain further understanding of many biological processes. The resulting chimera, termed J-SBD, was properly folded and had properties not present in the SBD alone. J-SBD behaved primarily as a monomer in all conditions tested and exhibited chaperone activity, as shown by aggregation protection and substrate binding assays, which revealed decreased binding to bis-ANS, a probe for hydrophobic patches. Collectively, our results suggest that Hsp40 binding to Hsp70 via the J-domain shifts the Hsp70 equilibrium towards the monomer state to expose hydrophobic sites prone to substrate accommodation.AbbreviationsBis-ANS (4,4’-Dianilino-1,1’-Binaphthyl-5,5’-Disulfonic Acid; CD, circular dichroism; Hsp, heat shock protein; J-SBD, chimeric polypeptide in which the J-domain of Hsp40 (at the N-terminus) is covalently attached to the substrate binding domain of Hsp70 (at the C-terminus) by a flexible linker; SBD: substrate binding domain of Hsp70.

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Necroptosis-blocking compound NBC1 targets heat shock protein 70 to inhibit MLKL polymerization and necroptosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1916503117 ◽

2020 ◽

Vol 117 (12) ◽

pp. 6521-6530 ◽

Cited By ~ 4

Author(s):

Andrea N. Johnston ◽

Yuyong Ma ◽

Hua Liu ◽

Shuzhen Liu ◽

Sarah Hanna-Addams ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Substrate Binding ◽

Kinase Domain ◽

Binding Domain ◽

Compound Libraries ◽

Cell Death Pathway ◽

Molecule Inhibitor ◽

Substrate Binding Domain

Necroptosis is a regulated necrotic cell death pathway involved in development and disease. Its signaling cascade results in the formation of disulfide bond-dependent amyloid-like polymers of mixed lineage kinase domain-like protein (MLKL), which mediate proinflammatory cell membrane disruption. We screened compound libraries provided by the National Cancer Institute and identified a small-molecule inhibitor of necroptosis named necroptosis-blocking compound 1 (NBC1). Biotin-labeled NBC1 specifically conjugates to heat shock protein Hsp70. NBC1 and PES-Cl, a known Hsp70 substrate-binding inhibitor, block the formation of MLKL polymers, but not MLKL tetramers in necroptosis-induced cells. In vitro,recombinant Hsp70 interacts with the N-terminal domain (NTD) of MLKL and promotes NTD polymerization, which has been shown to mediate the cell killing activity. Furthermore, the substrate-binding domain (SBD) of Hsp70 is sufficient to promote MLKL polymerization. NBC1 covalently conjugates cysteine 574 and cysteine 603 of the SBD to block its function. In addition, an SBD mutant with both cysteines mutated to serines loses its ability to promote MLKL polymerization. Interestingly, knockdown of Hsp70 in cells leads to MLKL destabilization, suggesting that MLKL might also be a client protein of Hsp70. In summary, using NBC1, an inhibitor of necroptosis, we identified Hsp70 as a molecular chaperone performing dual functions in necroptosis. It stabilizes MLKL protein under normal condition and promotes MLKL polymerization through its substrate-binding domain during necroptosis.

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Crystal Structure of the Stress-Inducible Human Heat Shock Protein 70 Substrate-Binding Domain in Complex with Peptide Substrate

PLoS ONE ◽

10.1371/journal.pone.0103518 ◽

2014 ◽

Vol 9 (7) ◽

pp. e103518 ◽

Cited By ~ 49

Author(s):

Pingfeng Zhang ◽

Julia I-Ju Leu ◽

Maureen E. Murphy ◽

Donna L. George ◽

Ronen Marmorstein

Keyword(s):

Crystal Structure ◽

Heat Shock ◽

Heat Shock Protein ◽

Heat Shock Protein 70 ◽

Substrate Binding ◽

Binding Domain ◽

Peptide Substrate ◽

Substrate Binding Domain ◽

Human Heat Shock Protein

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Studies on the effect of the J-domain on the substrate binding domain (SBD) of Hsp70 using a chimeric human J-SBD polypeptide

International Journal of Biological Macromolecules ◽

10.1016/j.ijbiomac.2018.11.130 ◽

2019 ◽

Vol 124 ◽

pp. 111-120 ◽

Cited By ~ 2

Author(s):

Ana O. Tiroli-Cepeda ◽

Thiago V. Seraphim ◽

Glaucia M.S. Pinheiro ◽

Denio E.P. Souto ◽

Lauro T. Kubota ◽

...

Keyword(s):

Substrate Binding ◽

Binding Domain ◽

J Domain ◽

Substrate Binding Domain

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A Histidine-rich and Cysteine-rich Metal-binding Domain at the C Terminus of Heat Shock Protein A fromHelicobacter pylori

Journal of Biological Chemistry ◽

10.1074/jbc.m800591200 ◽

2008 ◽

Vol 283 (22) ◽

pp. 15142-15151 ◽

Cited By ~ 78

Author(s):

Shujian Cun ◽

Hongyan Li ◽

Ruiguang Ge ◽

Marie C. M. Lin ◽

Hongzhe Sun

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Metal Binding ◽

Protein A ◽

Binding Domain ◽

C Terminus ◽

Metal Binding Domain

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The β6/β7 region of the Hsp70 substrate-binding domain mediates heat-shock response and prion propagation

Cellular and Molecular Life Sciences ◽

10.1007/s00018-017-2698-3 ◽

2017 ◽

Vol 75 (8) ◽

pp. 1445-1459 ◽

Cited By ~ 4

Author(s):

Linan Xu ◽

Weibin Gong ◽

Sarah A. Cusack ◽

Huiwen Wu ◽

Harriët M. Loovers ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Response ◽

Substrate Binding ◽

Binding Domain ◽

Shock Response ◽

Prion Propagation ◽

Substrate Binding Domain

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Defining the structure of the substrate-free state of the DnaK molecular chaperone

Biochemical Society Symposium ◽

10.1042/bss0680069 ◽

2001 ◽

Vol 68 ◽

pp. 69-82 ◽

Cited By ~ 3

Author(s):

Joanna F. Swain ◽

Renuka Sivendran ◽

Lila M. Gierasch

Keyword(s):

Binding Site ◽

Substrate Binding ◽

Binding Domain ◽

Full Length ◽

Free State ◽

Substrate Binding Site ◽

Terminal Domain ◽

C Terminus ◽

Ph Dependent ◽

Substrate Binding Domain

Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell. Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism. Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also. At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs. At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time. We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH. The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well. In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain.

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Abstract 1055: The 70 kDa Heat Shock Cognate Protein Plays A Novel Role In Myocardial Chemokine Expression And Cardiac Dysfunction Following Ischemia And Reperfusion Injury

Circulation ◽

10.1161/circ.116.suppl_16.ii_211 ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Ning Zou ◽

Lihua Ao ◽

Xiaoping Yang ◽

Xin Su ◽

David A Fullerton ◽

...

Keyword(s):

Heat Shock ◽

Inflammatory Response ◽

Cardiac Dysfunction ◽

Substrate Binding ◽

Binding Domain ◽

Chemokine Expression ◽

Heat Shock Cognate Protein ◽

Substrate Binding Domain ◽

Cognate Protein

Myocardial ischemia and reperfusion (I/R) causes the release of cellular proteins including heat shock proteins (HSPs). The inducible HSP70 induces cytokine production in monocytes and dendritic cells through Toll-like receptor 4 (TLR4) signaling. In evaluation of the role of HSPs in myocardial inflammatory response in a mouse heart global I/R model, we found that the 70 kDa heat shock cognate protein (HSC70), but not HSP70, was released into the extracellular space and the coronary effluent during I/R. These observations prompted us to hypothesize that HSC70 plays a novel role in postischemic myocardial inflammatory response and cardiac dysfunction. Methods: We subjected mouse hearts to global I/R (20 min/60 min) or perfusion using the Langendorff method. We examined: the effect of HSC70 antibody on myocardial chemokine expression and cardiac functional recovery following I/R, the effect of recombinant HSC70 on myocardial chemokine expression and cardiac function and the role of TLR4 and the HSC70 substrate-binding domain in the effect of HSC70 on the heart. Results: In comparison with non-immune IgG, anti-HSC70 reduced myocardial expression of KC and MCP-1 mRNAs and proteins following I/R. Moreover, treatment with anti-HSC70 improved postischemic cardiac functional recovery (66±5.4% of baseline vs. 28±5.1% of baseline in hearts treated with non-immune IgG, p<0.01). Recombinant HSC70 induced myocardial expression of KC and MCP-1 mRNAs and proteins and caused cardiac dysfunction (72±2.6% of baseline vs. 98±3.9% of baseline in perfusion controls, p<0.001) in hearts with competent TLR4 (C3H/HeN). Interestingly, these effects of HSC70 were abrogated in hearts with defective TLR4 (C3H/HeJ). The potency of HSC70 was completely lost in the absence of its substrate-binding domain. Conclusions: Taken together, our studies demonstrate, for the first time, that HSC70 plays an important role in the induction of myocardial chemokines and cardiac dysfunction during I/R. The effect of HSC70 is dependent on TLR4 and requires the presence of the substrate-binding domain. The results suggest that the release of HSC70 into the extracellular space elicits an inflammatory response and causes mechanic dysfunction in the heart.

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Structure of the substrate-binding domain of Plasmodium falciparum heat-shock protein 70-x

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x2001208x ◽

2020 ◽

Vol 76 (10) ◽

pp. 495-500

Author(s):

Julia Schmidt ◽

Ioannis Vakonakis

Keyword(s):

Plasmodium Falciparum ◽

Heat Shock ◽

Malaria Parasite ◽

Substrate Binding ◽

Binding Domain ◽

Crystallographic Structure ◽

Hydrophobic Peptide ◽

Febrile Episodes ◽

Substrate Binding Domain ◽

Human Hsp70

The malaria parasite Plasmodium falciparum extensively modifies erythrocytes that it invades by exporting a large complement of proteins to the host cell. Among these exported components is a single heat-shock 70 kDa class protein, PfHsp70-x, that supports the virulence and growth rate of the parasite during febrile episodes. The ATP-binding domain of PfHsp70-x has previously been resolved and showed the presence of potentially druggable epitopes that differ from those on human Hsp70 chaperones. Here, the crystallographic structure of the substrate-binding domain (SBD) of PfHsp70-x is presented in complex with a hydrophobic peptide. The PfHsp70-x SBD is shown to be highly similar to the counterpart from a human erythrocytic Hsp70 chaperone. The binding of substrate at the interface between β-sandwich and α-helical subdomains of this chaperone segment is also conserved between the malaria parasite and humans. It is hypothesized that the parasite may partly exploit human chaperones for intra-erythrocytic trafficking and maintenance of its exported proteome.

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