Fusion protein analysis reveals the precise regulation between Hsp70 and Hsp100 during protein disaggregation

Sayaka Hayashi,Yosuke Nakazaki,Kei Kagii,Hiromi Imamura,Yo-Hei Watanabe

doi:10.1038/s41598-017-08917-8

Sayaka Hayashi, Yosuke Nakazaki + Show 3 more

Open Access

https://doi.org/10.1038/s41598-017-08917-8

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Journal: Scientific Reports	Publication Date: Aug 17, 2017
Citations: 14	License type: open-access

Affiliation: Konan University, Kyoto University

Abstract

ClpB, a bacterial Hsp100, is a ring-shaped AAA+ chaperone that can reactivate aggregated proteins in cooperation with DnaK, a bacterial Hsp70, and its co-factors. ClpB subunits comprise two AAA+ modules with an interstitial rod-shaped M-domain. The M-domain regulates ClpB ATPase activity and interacts directly with the DnaK nucleotide-binding domain (NBD). Here, to clarify how these functions contribute to the disaggregation process, we constructed ClpB, DnaK, and aggregated YFP fusion proteins in various combinations. Notably, i) DnaK activates ClpB only when the DnaK substrate-binding domain (SBD) is in the closed conformation, affording high DnaK-peptide affinity; ii) although NBD alone can activate ClpB, SBD is required for disaggregation; and iii) tethering aggregated proteins to the activated ClpB obviates SBD requirements. These results indicate that DnaK activates ClpB only when the SBD tightly holds aggregated proteins adjacent to ClpB for effective disaggregation.

Highlights

Reactivation of damaged proteins is an important process for the survival of cells under stress conditions[1, 2]
The DnaK-dependent stimulation of ClpB ATPase activity was found, the stimulation required extremely high concentrations of DnaK and ClpB. Based on these results together with other related observations, the following model for the cooperation between DnaK and ClpB was proposed: i) DnaK binds aggregated protein; ii) ClpB binds DnaK through their respective MD and nucleotide-binding domain (NBD), stimulating the ClpB ATPase activity; iii) DnaK transfers the aggregated proteins to the activated ClpB; and iv) ClpB threads the aggregated proteins through its central pore to disaggregate them
ATP binding to the NBD induces the conformational change of DnaK to the open form, whereas ATP hydrolysis and phosphate release induce the change to the closed form

Summary

Introduction

Reactivation of damaged proteins is an important process for the survival of cells under stress conditions[1, 2]. In the ATP bound state, SBDα detaches SBDβ and the substrate-binding site becomes exposed to the solvent[12, 13] In such open form, the SBD rapidly binds and releases denatured proteins with relatively low affinity[14, 15]. The DnaK-dependent stimulation of ClpB ATPase activity was found, the stimulation required extremely high concentrations of DnaK and ClpB Based on these results together with other related observations, the following model for the cooperation between DnaK and ClpB was proposed: i) DnaK binds aggregated protein; ii) ClpB binds DnaK through their respective MD and NBD, stimulating the ClpB ATPase activity; iii) DnaK transfers the aggregated proteins to the activated ClpB; and iv) ClpB threads the aggregated proteins through its central pore to disaggregate them. Precise investigation of these issues has been difficult primarily owing to the low affinity between DnaK and ClpB (Kd ≈ 25 μM)[41]

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Results

Conclusion