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. 2016 Sep 16;291(38):19848-57.
doi: 10.1074/jbc.M116.742502. Epub 2016 Jul 29.

Non-canonical Interactions between Heat Shock Cognate Protein 70 (Hsc70) and Bcl2-associated Anthanogene (BAG) Co-Chaperones Are Important for Client Release

Jennifer N Rauch  1 , Erik R P Zuiderweg  1 , Jason E Gestwicki  2
Affiliations

Non-canonical Interactions between Heat Shock Cognate Protein 70 (Hsc70) and Bcl2-associated Anthanogene (BAG) Co-Chaperones Are Important for Client Release

Jennifer N Rauch et al. J Biol Chem. .

Abstract

Heat shock cognate protein 70 (Hsc70) regulates protein homeostasis through its reversible interactions with client proteins. Hsc70 has two major domains: a nucleotide-binding domain (NBD), that hydrolyzes ATP, and a substrate-binding domain (SBD), where clients are bound. Members of the BAG family of co-chaperones, including Bag1 and Bag3, are known to accelerate release of both ADP and client from Hsc70. The release of nucleotide is known to be mediated by interactions between the conserved BAG domain and the Hsc70 NBD. However, less is known about the regions required for client release, and it is often assumed that this activity also requires the BAG domain. It is important to better understand this step because it determines how long clients remain in the inactive, bound state. Here, we report the surprising observation that truncated versions of either human Bag1 or Bag3, comprised only the BAG domain, promoted rapid release of nucleotide, but not client, in vitro Rather, we found that a non-canonical interaction between Bag1/3 and the Hsc70 SBD is sufficient for accelerating this step. Moreover, client release did not seem to require the BAG domain or Hsc70 NBD. These results suggest that Bag1 and Bag3 control the stability of the Hsc70-client complex using at least two distinct protein-protein contacts, providing a previously under-appreciated layer of molecular regulation in the human Hsc70 system.

Keywords: 70 kilodalton heat shock protein (Hsp70); molecular chaperone; nuclear magnetic resonance (NMR); protein complex; protein-protein interaction.

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Figures

FIGURE 1.
FIGURE 1.
Isolated BAG domains promote release of nucleotide, but not client, from Hsc70. A, partial domain architecture of human Bag1 and Bag3, highlighting the location of the BAG domain. Some known domains are not indicated for clarity. Bag1C and Bag3C are the isolated BAG domains. B, full-length Bag1/3, but not Bag1C or Bag3C, strongly promote release of a fluorescent client from Hsc70. The results are the average of four independent experiments performed in triplicate each. The error bars represent S.D. All of the NEFs were able to promote nucleotide release, as previously reported (see text).
FIGURE 2.
FIGURE 2.
Design of point mutations in Bag1 and Bag3 that disrupt binding of the BAG domain to Hsc70. A, binding interface between Bag1/3 and Hsc70's NBD, highlighting the polar contacts. B, CD spectra of Bag1 and Bag3 proteins, showing that the mutations do not disrupt the overall structure. C, mutations in Bag1 or Bag3 interrupt binding to the Hsc70 NBD, as measured by FCPIA. D, mutations in Bag1/3 disrupt effects on the Hsc70 ATPase activity (left) and the refolding of denatured firefly luciferase (right). NEFs, such as Bag1 and Bag3, typically stimulate ATPase and luciferase refolding activity at low concentrations and then inhibit at higher ratios. In C and D, the results are shown as the average of at least three experiments performed in triplicate. The error bars represent S.D.
FIGURE 3.
FIGURE 3.
Mutation or deletion of the BAG domain blocks the ability to promote ADP release, but not client release. A, Bag1/3 mutants have decreased ability to release nucleotide from Hsp70. B, Bag1/3 mutants have normal activity in the client release assay. C, domain structure of the Bag3ΔBAG construct used in these studies is shown. D, deletion of the BAG domain from Bag3 has no effect on client release. E, deletion of the BAG domain from Bag3 abolishes nucleotide release. For all experiments, the results are the average of at least three experiments performed in triplicate each, and the error bars represent S.D.
FIGURE 4.
FIGURE 4.
The non-canonical interaction between Bag3 and Hsp72 occurs in the SBD. A, truncations of Hsc70 used in this study. B, client tracer interacts with both full-length Hsc70 and Hsc70SBD. C, Bag1, 3, double mutants and Bag3ΔBAG compete with client for binding to the Hsc70SBD. Results are the average of experiments performed in triplicate. Error is S.E. D, 600 MHz 13C-1H HSQC spectra of 11 μm 13C 15N Hsc70 (395–508) showing binding to a model client (NRLLLTG), to Bag3ΔBAG and to Bag3C. In each overlay, the apo-protein spectrum is in blue, and the spectrum of the Hsc70 SBDβ domain in the presence of ligand is shown in red. The model client and Bag3ΔBAG cause chemical shift perturbations and appearances of new resonances in slow exchange at ∼1:1, whereas Bag3C only causes minor changes at ∼3:1. E, model for the bi-dentate mechanism of human Bag1 and Bag3. The identity and location of the SBD-binding site is not yet clear (see supplemental Fig. S1).
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References

    1. Mayer M. P., and Bukau B. (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol. Life Sci. 62, 670–684 - PMC - PubMed
    1. Hartl F. U., Bracher A., and Hayer-Hartl M. (2011) Molecular chaperones in protein folding and proteostasis. Nature 475, 324–332 - PubMed
    1. Assimon V. A., Gillies A. T., Rauch J. N., and Gestwicki J. E. (2013) Hsp70 protein complexes as drug targets. Curr. Pharm. Des. 19, 404–417 - PMC - PubMed
    1. Rampelt H., Mayer M. P., and Bukau B. (2011) Nucleotide exchange factors for Hsp70 chaperones. Methods Mol. Biol. 787, 83–91 - PubMed
    1. Zuiderweg E. R., Bertelsen E. B., Rousaki A., Mayer M. P., Gestwicki J. E., and Ahmad A.. (2013) Allostery in the Hsp70 chaperone proteins. Topics in Current Chemistry 328, 99–153 - PMC - PubMed

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