The chaperone function of ClpB from Thermus thermophilus depends on allosteric interactions of its two ATP-binding sites

Schlee, Sandra; Groemping, Yvonne; Herde, Petra; Seidel, R.; Reinstein, Jochen

doi:10.1006/jmbi.2001.4455

Cited by 84 publications

(158 citation statements)

References 34 publications

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“…In prokaryotes, as we have demonstrated for chaperones from a thermophilic eubacteria, Thermus thermophilus, heatdamaged proteins were rescued by the cooperative chaperone functions of ClpB (bacterial HSP104), DnaK (bacterial HSP70), DnaJ, and GrpE (4,11). ClpB forms a homo-hexameric complex (580 kDa) as an active species and needs ATP hydrolysis for the function (12)(13)(14)(15)(16)(17). DnaK also has ATPase activity and works with DnaJ and GrpE (termed the DnaK set) (2).…”

mentioning

confidence: 98%

“…The stable K⅐J has been reported only for DnaK and DnaJ from T. thermophilus, but the chaperone function of K⅐J has been studied with a general interest because the current model for the mechanism of DnaK function assumes only transient but not stable interaction between DnaK and DnaJ (4,11,16,19,23). Related to this contention, the chaperone activity of uncomplexed TDnaK and TDnaJ (termed KϩJ) has been reported (14,23,24), but the difference from that of K⅐J is not clear. Here, we carefully compared chaperone activities of the K⅐J set (K⅐J plus TGrpE) and those of the KϩJ set (KϩJ plus TGrpE).…”

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confidence: 99%

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Trigonal DnaK-DnaJ Complex Versus Free DnaK and DnaJ

Watanabe¹,

Yoshida

2004

Journal of Biological Chemistry

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DnaK from Thermus thermophilus (TDnaK) is unique because significant fractions of cellular TDnaK exist as a trigonal K⅐J complex that consists of three copies each of TDnaK, TDnaJ, and an assembly factor TDafA. Here, chaperone functions of the K⅐J complex and free TDnaK plus free TDnaJ (K؉J) were compared. Substrate proteins were completely denatured at 72-73°C or 89°C in the absence or the presence of K⅐J complex or K؉J and were subsequently incubated at a moderate temperature of 55°C. TGrpE and ATP were always included in the K⅐J complex and K؉J, and TClpB was supplemented at 55°C. At 72-73°C, both the K⅐J complex and K؉J suppressed heat aggregation of substrate proteins. During the next incubation at 55°C, K؉J, assisted by TClpB, was able to disaggregate the heat aggregates and efficiently reactivate activities of the proteins, whereas the K⅐J complex was not; it reactivated only the soluble inactivated proteins. When substrate proteins were heated to 89°C, both the K⅐J complex and K؉J were no longer able to prevent heat aggregation, and because of selective, irreversible denaturation of TDafA the K⅐J complex dissociated into K؉J, which then exhibited disaggregation activity during the next incubation at 55°C. Thus, TClpB-assisted disaggregation activity belongs only to K؉J, and TDafA is a potential thermosensor for converting the K⅐J complex to K؉J in response to heat stress.

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confidence: 98%

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confidence: 99%

Trigonal DnaK-DnaJ Complex Versus Free DnaK and DnaJ

Watanabe¹,

Yoshida

2004

Journal of Biological Chemistry

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“…Previous biochemical studies have revealed that both Hsp104 and ClpB show cooperativity between nucleotide binding sites that controls their ATPase activity (18,19). Mutations at any of the NBDs that inhibit their ATPase activity also impair chaperone activity, indicating that ATP hydrolysis at both NBDs is required for productive substrate handling (20).…”

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confidence: 99%

Allosteric Communication between the Nucleotide Binding Domains of Caseinolytic Peptidase B

Fernández-Higuero

Acebrón

Taneva

et al. 2011

Journal of Biological Chemistry

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ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood. We have studied herein the affinity of each of the NBDs for nucleotides in WT ClpB and protein variants in which one or both sites are mutated to selectively impair nucleotide binding or hydrolysis. Our data show that the affinity of NBD2 for nucleotides (K d ‫؍‬ 3-7 M) is significantly higher than that of NBD1. Interestingly, the affinity of NBD1 depends on nucleotide binding to NBD2. Binding of ATP, but not ADP, to NBD2 increases the affinity of NBD1 (the K d decreases from ≈160 -300 to 50 -60 M) for the corresponding nucleotide. Moreover, filling of the NBD2 ring with ATP allows the cooperative binding of this nucleotide and substrates to the NBD1 ring. Data also suggest that a minimum of four subunits cooperate to bind and reactivate two different aggregated protein substrates.

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“…The steady-state ATPase activity of ClpB NBD2 variants was measured in a coupled colorimetric assay essentially as described previously (Schlee et al, 2001;Beinker et al, 2005). The measurements were performed at 25 C using a Jasco V-650 spectrophotometer (Jasco Germany GmbH).…”

Section: Steady-state Atpase Assaymentioning

confidence: 99%

“…It has previously been shown that this mutation causes severe defects in both nucleotide binding and ATPase activity (Schlee et al, 2001). To crystallize the mutant ClpB variant NBD2 K601Q, we supplemented the crystallization cocktail with the fluorescently labelled nucleotide MANT-dADP, which has been routinely used for nucleotidebinding studies of ClpB and is known to bind more than 100-fold more strongly to NBD2 than unlabelled ADP (Werbeck et al, 2009).…”

Section: Implications From a Clpbmentioning

confidence: 99%

Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

Zeymer

Barends

Werbeck

et al. 2014

Acta Cryst D Biol Crystallogr

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ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotidebinding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically. The results provide insights into nucleotide sensing, explaining how the conserved sensor 2 motif contributes to the discrimination between ADP and ATP binding. Furthermore, the role of a conserved active-site arginine (Arg621), which controls binding of the essential Mg 2+ ion, is described. Finally, a hypothesis is presented as to how the ATPase activity is regulated by a conformational switch that involves the essential Walker A lysine. In the proposed model, an unusual side-chain conformation of this highly conserved residue stabilizes a catalytically inactive state, thereby avoiding unnecessary ATP hydrolysis.

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The chaperone function of ClpB from Thermus thermophilus depends on allosteric interactions of its two ATP-binding sites

Cited by 84 publications

References 34 publications

Trigonal DnaK-DnaJ Complex Versus Free DnaK and DnaJ

Trigonal DnaK-DnaJ Complex Versus Free DnaK and DnaJ

Allosteric Communication between the Nucleotide Binding Domains of Caseinolytic Peptidase B

Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

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