Functional characterization of the DnaK chaperone system from the archaeon <i>Methanothermobacter thermautotrophicus</i><i>ΔH</i>

Popp, Simone; Reinstein, Jochen

doi:10.1016/j.febslet.2008.12.062

Cited by 8 publications

(8 citation statements)

References 30 publications

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“…Mortalin likely presents similar thermal induced unfolding in a number of events, but we did not observe the third transition by CD 222 nm . Interestingly, E. coli DnaK thermal induced unfolding followed by CD 222nm also unfolding through two transitions with similar Tms to those observed for mortalin [ 59 – 61 ].…”

Section: Resultsmentioning

confidence: 57%

Human Mitochondrial Hsp70 (Mortalin): Shedding Light on ATPase Activity, Interaction with Adenosine Nucleotides, Solution Structure and Domain Organization

et al. 2015

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The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-encoded proteins. The in vivo deregulation of mortalin expression and/or function has been correlated with age-related diseases and certain cancers due to its interaction with the p53 protein. In spite of its critical biological roles, structural and functional studies on mortalin are limited by its insoluble recombinant production. This study provides the first report of the production of folded and soluble recombinant mortalin when co-expressed with the human Hsp70-escort protein 1, but it is still likely prone to self-association. The monomeric fraction of mortalin presented a slightly elongated shape and basal ATPase activity that is higher than that of its cytoplasmic counterpart Hsp70-1A, suggesting that it was obtained in the functional state. Through small angle X-ray scattering, we assessed the low-resolution structural model of monomeric mortalin that is characterized by an elongated shape. This model adequately accommodated high resolution structures of Hsp70 domains indicating its quality. We also observed that mortalin interacts with adenosine nucleotides with high affinity. Thermally induced unfolding experiments indicated that mortalin is formed by at least two domains and that the transition is sensitive to the presence of adenosine nucleotides and that this process is dependent on the presence of Mg2+ ions. Interestingly, the thermal-induced unfolding assays of mortalin suggested the presence of an aggregation/association event, which was not observed for human Hsp70-1A, and this finding may explain its natural tendency for in vivo aggregation. Our study may contribute to the structural understanding of mortalin as well as to contribute for its recombinant production for antitumor compound screenings.

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Section: Resultsmentioning

confidence: 57%

Human Mitochondrial Hsp70 (Mortalin): Shedding Light on ATPase Activity, Interaction with Adenosine Nucleotides, Solution Structure and Domain Organization

et al. 2015

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“…In contrast to DnaJ from Escherichia coli or Methanothermobacter thermautotrophicus ÁH (Laufen et al, 1999;Popp & Reinstein, 2009;Russell et al, 1999), DnaJ from Thermus thermophilus stimulates the ATPase activity of DnaK only very weakly (Groemping et al, 2001;Klostermeier et al, 1999;Motohashi et al, 1997). Nevertheless, DnaJ is essential and highly effective in preventing aggregation of challenged protein-folding intermediates as part of the T. thermophilus DnaK-DnaJ-GrpE chaperone system.…”

Section: Introductionmentioning

confidence: 99%

Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

Barends

Brosi

Steinmetz

et al. 2013

Acta Crystallogr D Biol Crystallogr

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Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.

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“…CeHsc70 alone was not able to refold luciferase, while addition of DNJ-13 resulted in refolding activity (Figure 6A). Addition of substoichiometric amounts of BAG-1 increased the refolding efficiency further (Figure 6A and 6B), but higher concentrations of BAG-1 reduced it to baseline levels (Figure 6B) revealing a clear optimum of NEF concentrations similar to the prokaryotic system [56]. We were interested, whether ATP hydrolysis followed the same trend.…”

Section: Resultsmentioning

confidence: 95%

The Lid Domain of Caenorhabditis elegans Hsc70 Influences ATP Turnover, Cofactor Binding and Protein Folding Activity

Sun¹,

Edelmann²,

Kaiser³

et al. 2012

PLoS ONE

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Hsc70 is a conserved ATP-dependent molecular chaperone, which utilizes the energy of ATP hydrolysis to alter the folding state of its client proteins. In contrast to the Hsc70 systems of bacteria, yeast and humans, the Hsc70 system of C. elegans (CeHsc70) has not been studied to date.We find that CeHsc70 is characterized by a high ATP turnover rate and limited by post-hydrolysis nucleotide exchange. This rate-limiting step is defined by the helical lid domain at the C-terminus. A certain truncation in this domain (CeHsc70-Δ545) reduces the turnover rate and renders the hydrolysis step rate-limiting. The helical lid domain also affects cofactor affinities as the lidless mutant CeHsc70-Δ512 binds more strongly to DNJ-13, forming large protein complexes in the presence of ATP. Despite preserving the ability to hydrolyze ATP and interact with its cofactors DNJ-13 and BAG-1, the truncation of the helical lid domain leads to the loss of all protein folding activity, highlighting the requirement of this domain for the functionality of the nematode's Hsc70 protein.

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Functional characterization of the DnaK chaperone system from the archaeon Methanothermobacter thermautotrophicusΔH

Cited by 8 publications

References 30 publications

Human Mitochondrial Hsp70 (Mortalin): Shedding Light on ATPase Activity, Interaction with Adenosine Nucleotides, Solution Structure and Domain Organization

Human Mitochondrial Hsp70 (Mortalin): Shedding Light on ATPase Activity, Interaction with Adenosine Nucleotides, Solution Structure and Domain Organization

Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

The Lid Domain of Caenorhabditis elegans Hsc70 Influences ATP Turnover, Cofactor Binding and Protein Folding Activity

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