Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. I. Kinetic analyses of active site mutants.

Wilbanks, Sigurd M.; DeLuca-Flaherty, Camille; McKay, David

doi:10.1016/s0021-9258(18)99960-6

Cited by 97 publications

(38 citation statements)

References 21 publications

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“…Replacements of Pro 143 and Glu 171 reduced the basal ATP hydrolysis rate of DnaK by one to two orders of magnitude, and the g-phosphate cleavage rate of the mutant proteins was not stimulated by DnaJ, but a stimulation by substrate or a combination of DnaJ and substrate could be observed (Figures 2B and 2C). While the overall decrease of the ATPase rate by a replacement of Glu 171 is easily explained by its function in coordination of the catalytically important Mg 2+ , consistent with earlier findings for Hsc70 (Wilbanks et al, 1994), the effects of the exchange of Pro 143 cannot be explained through a direct role in catalysis but instead are consistent with the hypothesis that Pro 143 is important for the positioning of the catalytic residue Lys 70 (Lys 71 in Hsc70). The allosteric stimulation of the ATPase activity, however, seems to be partially functional in all variants tested with replacements in these two positions.…”

Section: Resultssupporting

confidence: 87%

Allosteric Regulation of Hsp70 Chaperones by a Proline Switch

2006

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Crucial to the function of Hsp70 chaperones is the nucleotide-regulated transition between two conformational states, the ATP bound state with high association and dissociation rates for substrates and the ADP bound state with two and three orders of magnitude lower association and dissociation rates. The spontaneous transition between the two states is extremely slow, indicating a high energy barrier for the switch that regulates the transition. Here we provide evidence that a universally conserved proline in the ATPase domain constitutes the switch that assumes alternate conformations in response to ATP binding and hydrolysis. The conformation of the proline, acting through an invariant arginine as relay, determines and stabilizes the opened and closed conformation of the substrate binding domain and thereby regulates the chaperone activity of Hsp70.

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Section: Resultssupporting

confidence: 87%

Allosteric Regulation of Hsp70 Chaperones by a Proline Switch

2006

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“…The availability of ATP is thus a time‐critical variable in the chaperone‐mediated suppression of Htt amyloid formation. To further analyze the ATP requirements, we created point mutations in HSP‐1 and HSP‐110 that abrogate or reduce ATP‐hydrolysis rates in the respective chaperone (Wilbanks et al , ; O'Brien et al , ). We confirmed the diminished ATPase activity in ATPase assays (Fig EV1G) and demonstrate that the point mutations do not affect the fold/secondary structure of the proteins as assessed by circular dichroism (CD) analysis (Fig EV1E and F).…”

Section: Resultsmentioning

confidence: 99%

Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex

et al. 2017

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Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin gene (). Molecular chaperones have been implicated in suppressing or delaying the aggregation of mutant Htt. Using and assays, we have identified a trimeric chaperone complex (Hsc70, Hsp110, and J-protein) that completely suppresses fibrilization of HttExon1Q The composition of this chaperone complex is variable as recruitment of different chaperone family members forms distinct functional complexes. The trimeric chaperone complex is also able to resolubilize Htt fibrils. We confirmed the biological significance of these findings in HD patient-derived neural cells and on an organismal level in Among the proteins in this chaperone complex, the J-protein is the concentration-limiting factor. The single overexpression of DNAJB1 in HEK293T cells is sufficient to profoundly reduce HttExon1Q aggregation and represents a target of future therapeutic avenues for HD.

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“…Human Hsp40 was expressed from the plasmid pET-Hsp40 in E.coli BL21 (DE3) cells and purified as described by Minami et al (1996). BL21 (DE3) cells were also used to express the ATPase domain of bovine Hsc70 from a corresponding pET plasmid, and subsequent purification of the ATPase domain was performed according to Willbanks et al (1994). Protein concentrations were determined using Bradford assay reagent (Bio-Rad) with bovine γ-globulin as the standard.…”

Section: Methodsmentioning

confidence: 99%

GrpE-like regulation of the Hsc70 chaperone by the anti-apoptotic protein BAG-1

Hohfeld¹,

Jentsch²

1998

The EMBO Journal

108

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The BAG-1 protein appears to inhibit cell death by binding to Bcl-2, the Raf-1 protein kinase, and certain growth factor receptors, but the mechanism of inhibition remains enigmatic. BAG-1 also interacts with several steroid hormone receptors which require the molecular chaperones Hsc70 and Hsp90 for activation. Here we show that BAG-1 is a regulator of the Hsc70 chaperone. BAG-1 binds to the ATPase domain of Hsc70 and, in cooperation with Hsp40, stimulates Hsc70's steady-state ATP hydrolysis activity~40-fold. Similar to the action of the GrpE protein on bacterial Hsp70, BAG-1 accelerates the release of ADP from Hsc70. Thus, BAG-1 regulates the Hsc70 ATPase in a manner contrary to the Hsc70-interacting protein Hip, which stabilizes the ADP-bound state. Intriguingly, BAG-1 and Hip compete in binding to the ATPase domain of Hsc70. Our results reveal an unexpected diversity in the regulation of Hsc70 and raise the possibility that the observed anti-apoptotic function of BAG-1 may be exerted through a modulation of the chaperone activity of Hsc70 on specific protein folding and maturation pathways.

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Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. I. Kinetic analyses of active site mutants.

Cited by 97 publications

References 21 publications

Allosteric Regulation of Hsp70 Chaperones by a Proline Switch

Allosteric Regulation of Hsp70 Chaperones by a Proline Switch

Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex

GrpE-like regulation of the Hsc70 chaperone by the anti-apoptotic protein BAG-1

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