The Second Step of ATP Binding to DnaK Induces Peptide Release

Theyssen, Holger; Schuster, Hans-Peter; Packschies, Lars; Bukau, Bernd; Reinstein, J.

doi:10.1006/jmbi.1996.0606

Cited by 210 publications

(277 citation statements)

References 31 publications

(47 reference statements)

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“…4 B-D), the extended conformation of rhodanese disappears with a rate constant of (2.5 ± 0.4) 10 −3 s −1 (Table S1). This rate is in a similar range as previously determined off-rates for substrates from ADP-bound DnaK (38,47,48). After reaching equilibrium, the transfer efficiency histograms are indistinguishable from those of the DnaJ-rhodanese complex, suggesting complete dissociation of DnaK (compare Fig.…”

Section: Dynamics Of Complex Formation From Microfluidicsupporting

confidence: 86%

“…The third component acting together with DnaJ and DnaK has not been considered so far: the nucleotide exchange factor GrpE (53). GrpE catalyzes the exchange of ADP with ATP in DnaK (54) and thus accelerates the nonequilibrium association/dissociation cycle of DnaK with the substrate protein (47,55). As a result, the addition of GrpE to the rhodaneseDnaK complexes leads to a faster disappearance of the expanded rhodanese population (Fig.…”

Section: Dynamics Of Complex Formation From Microfluidicmentioning

confidence: 99%

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Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

Kellner

Hofmann

Barducci

et al. 2014

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

108

118

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Molecular chaperones are an essential part of the machinery that avoids protein aggregation and misfolding in vivo. However, understanding the molecular basis of how chaperones prevent such undesirable interactions requires the conformational changes within substrate proteins to be probed during chaperone action. Here we use single-molecule fluorescence spectroscopy to investigate how the DnaJ-DnaK chaperone system alters the conformational distribution of the denatured substrate protein rhodanese. We find that in a first step the ATP-independent binding of DnaJ to denatured rhodanese results in a compact denatured ensemble of the substrate protein. The following ATP-dependent binding of multiple DnaK molecules, however, leads to a surprisingly large expansion of denatured rhodanese. Molecular simulations indicate that hard-core repulsion between the multiple DnaK molecules provides the underlying mechanism for disrupting even strong interactions within the substrate protein and preparing it for processing by downstream chaperone systems.Hsp70 | Hsp40 | Förster resonance energy transfer | protein folding | FRET

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Section: Dynamics Of Complex Formation From Microfluidicsupporting

confidence: 86%

Section: Dynamics Of Complex Formation From Microfluidicmentioning

confidence: 99%

Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein

Kellner

Hofmann

Barducci

et al. 2014

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

108

118

View full text Add to dashboard Cite

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“…Fluorescence anisotropy measurements of fp5 incubated with DnaK were made essentially as described previously (12) but in the presence of 1 mM ADP. Kinetic measurements were made using a Bio-Logic stopped-flow device similarly as described previously (23) except that fp5 was used as a substrate with excitation at 494 nm and detection at 517 nm. In control experiments, mixing with buffer instead of ATP showed no increase in fp5 fluorescence over time, and mixing with saturating concentrations of both ATP and p5 gave the same kinetic trace as addition of ATP alone, indicating that release of fp5 was both ATP-dependent and complete.…”

Section: Methodsmentioning

confidence: 99%

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

Smock

Blackburn

Gierasch

2011

Journal of Biological Chemistry

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The 70-kDa heat shock proteins (Hsp70s) function as molecular chaperones through the allosteric coupling of their nucleotide-and substrate-binding domains, the structures of which are highly conserved. In contrast, the roles of the poorly structured, variable length C-terminal regions present on Hsp70s remain unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD tetrapeptide sequence associates with co-chaperones via binding to tetratricopeptide repeat domains. It is not known whether this is the only function for this region in eukaryotic Hsp70s and what roles this region performs in Hsp70s that do not form complexes with tetratricopeptide repeat domains. We compared C-terminal sequences of 730 Hsp70 family members and identified a novel conservation pattern in a diverse subset of 165 bacterial and organellar Hsp70s. Mutation of conserved C-terminal sequence in DnaK, the predominant Hsp70 in Escherichia coli, results in significant impairment of its protein refolding activity in vitro without affecting interdomain allostery, interaction with co-chaperones DnaJ and GrpE, or the binding of a peptide substrate, defying classical explanations for the chaperoning mechanism of Hsp70. Moreover, mutation of specific conserved sites within the DnaK C terminus reduces the capacity of the cell to withstand stresses on protein folding caused by elevated temperature or the absence of other chaperones. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.The ubiquitously distributed Hsp70 family of molecular chaperones shepherd newly synthesized polypeptide chains, protect cells from stress-induced protein aggregation, assist in protein translocation across membranes, and regulate assembly and disassembly of macromolecular complexes. These physiological functions are accomplished by a two-domain allosteric mechanism in which cycles of ATP binding and hydrolysis in the N-terminal nucleotide-binding domain (NBD) 2 control the binding and release of hydrophobic polypeptide segments in the substrate-binding domain (SBD) (1-3). Although we have gained an increasingly detailed picture of this allosteric transition in Hsp70 chaperones and modes of substrate interaction (4 -7), it is striking that there is much less known about the function of the extreme C-terminal unstructured region (Fig. 1). Binding of the C-terminal region of mammalian Hsc70 to substrate was suggested in earlier work (8, 9), and more recently, the C-terminal segment of eukaryotic cytoplasmic Hsp70s was found to contain a conserved tetratricopeptide repeat (TPR) domain interaction motif that mediates binding with Chip, Hip, or Hop co-chaperones that facilitate the assembly of a variety of Hsp70 complexes (3). Even though bacteria lack homologs of these Hsp70-interacting TPR domain proteins, many, including the extensively characterized E. coli Hsp70 DnaK, have a disord...

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“…In all of these processes, DnaK is thought to interact transiently with unraveled segments of partially unfolded or denatured protein substrates. Studies have shown that the reversible switching from a high affinity conformation, which binds substrate tightly, to a low affinity conformation, which binds substrate weakly, is the hallmark of DnaK activity (7)(8)(9)(10)(11). Such a mechanism is shown in Scheme 1, where ADP⅐DnaK⅐P and ATP⅐DnaK* are the high and low affinity states, respectively, and E and J are GrpE and DnaJ, respectively.…”

mentioning

confidence: 99%