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The Holdase Function of Escherichia Coli Hsp70 (Dnak) Chaperone

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The Holdase Function of Escherichia Coli Hsp70 (Dnak) Chaperone bioRxiv preprint doi: https://doi.org/10.1101/305854; this version posted April 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The holdase function of Escherichia coli Hsp70 (DnaK) chaperone Ricksen S. Winardhi,1, 2, ∗ Qingnan Tang,1, 2, ∗ Huijuan You,2 Michael Sheetz,2 and Jie Yan1, 2, 3, y 1Department of Physics, National University of Singapore, Singapore 117542 2Mechanobiology Institute, National University of Singapore, Singapore 117411 3Centre for Bioimaging Sciences, National University of Singapore, Singapore 117546 (Dated: April 21, 2018) In Escherichia coli, the DnaK/DnaJ/GrpE system plays a critical role in mediating protein re- folding and buffering against protein aggregation due to environmental stress. The underlying mechanism remains unclear. In this work, we probe the activity of DnaK/DnaJ/GrpE system with single-molecule protein refolding assay using tandem repeats of titin immunoglobulin 27 (I27)8. We provide direct evidence that DnaK in apo- and ADP-bound state is predominantly a holdase, which kinetically stabilizes the polyprotein in its unfolded form. Binding of ATP relieves DnaK's holding, allowing protein refolding. The presence of co-chaperone DnaJ and GrpE modulates this holding- release switching, possibly by altering DnaK's nucleotide state. Our findings thus provide important insights to the molecular mechanism of DnaK/DnaJ/GrpE system. INTRODUCTION Hsp70 is of special interest due to its implication in var- ious neurodegenerative diseases, as many of these are In virtually all living organisms, proteins are synthe- caused by accumulation of aggregates/misfolded proteins sized by a molecular machine called ribosome by sequen- [3]. Hsp70 also plays another critical role in regulat- tial addition of amino acids that forms a polypeptide ing cell signaling, including type II diabetes and cancer- chain. The nascent polypeptide chain released from the related signaling pathways [4]. Studies on Hsp70 sys- ribosome then needs to fold into its native protein struc- tem mainly focus on DnaK, which is the major bac- ture. This is a complex and vital process that often re- terial Hsp70 in E. coli, as a model. DnaK comprises quires the assistance of other proteins, broadly termed as of two domains: N-terminal nucleotide-binding domain chaperones. There are many different families of chaper- (NBD) and C-terminal substrate-binding domain (SBD), ones, which are categorized according to their molecular connected via a flexible and highly conserved linker [5]. weight, generally prefixed by the term `Hsp' as histori- The SBD contains a β-sheet peptide binding groove/cleft cally these proteins are named `heat shock protein' due (SBD-β) for substrate binding, and an α-helical lid to their high expression under stress condition such as (SBD-α) for substrate locking. heat shock [1]. The NBD is responsible for ATPase activity, which is Each of the chaperone family serves a different func- important in switching the SBD conformation between tion in the general housekeeping of proteins in cells, and open and closed state, i.e. whether the SBD-α is de- they often works in synergy with each other. For exam- tached or attached to the SBD-β. In the apo-state and ple, Hsp70 family of proteins assists client folding in its ADP-bound state, the NBD is undocked from the SBD, early stage by binding to the nascent polypeptide chain and the SBD-α is pressed against the SBD-β [5], while on the ribosomes, while Hsp90 assists during late stage in the ATP-bound state the NBD is docked to the SBD folding. In addition, proteins that are damaged due to with SBD-α separated away from SBD-β [6, 7]. Thus, environmental stress need to be either repaired or de- ATP hydrolysis cycle dictates the conformational state of graded. Hsp60 can help to refold denatured proteins, as the SBD in DnaK to control binding activity to peptide well as promote de novo folding of polypeptides. Another substrate. Recently, the importance of SBD-β allostery class of chaperones, Hsp100, utilizes energy from ATP to in actively controlling substrate binding and release was actively unfold and disaggregate misfolded protein struc- elucidated [8]. As such, substrate affinity depends on tures and aggregates. In short, the complex network of whether the conformation of SBD is in the open or closed chaperones serves many functions: assisting folding of state. Substrate binding can also alter DnaK conforma- newly synthesized or denatured proteins, preventing ag- tional state [9, 10], which has been suggested to stimu- gregation, unfolding of protein aggregates, all of which late ATPase activity observed upon substrate binding up are crucial in order to maintain proteome integrity and to 25-fold [11{13]. Taken together, the substrate binding protein homeostasis [2]. activity depends on the complex interplay of multiple fac- Among the various family of molecular chaperones, tors: SBD-β dynamics, SBD-α lid opening/closure, pep- tide binding, and the nucleotide-state that tightly control allostericity. ∗ The authors wish it to be known that, in their opinion, the first In addition to its conformational dynamics, the ac- two authors should be regarded as joint First Authors tivity of DnaK also depends on other factors, such as y Corresponding author e-mail: [email protected] its oligomerization activity [14] and its interaction with bioRxiv preprint doi: https://doi.org/10.1101/305854; this version posted April 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 2 other chaperones and co-chaperones [15]. In the ATP- inteference from the commonly used denaturants, 2) pro- bound state of DnaK (without peptide), the intrinsic tein substrate can be reliably unfolded and refolded over ATPase activity of DnaK is very slow [0.02 min-1 at a long period of time, and 3) single-protein sensitivity can 25 ◦C] [11, 16, 17]. Assistance of DnaJ, which is bac- be achieved. We find that DnaK in the apo- and ADP- terial Hsp40 protein, can accelerate the rate ATP hy- bound state can serve as a very effective holdase. Upon drolysis by a maximum of 15,000-fold at 5 ◦C [11], and binding of ATP, DnaK holdase activity is relieved to al- induces the α-helical lid to close and lock the substrate low for protein refolding, while addition of DnaJ in the in the SBD. Following ATP hydrolysis, a nucleotide presence of ATP converts DnaK back to a holdase. We exchange factor (NEF), GrpE, helps to facilitate nu- also find that further addition of GrpE can relieve this cleotide exchange by stimulating ADP release by 5,000- DnaJ-dependent holdase activity of DnaK in the presence fold [18]. Together with its co-chaperones, DnaK forms of ATP. the DnaK/DnaJ/GrpE system for efficient chaperoning activity in E. coli. DnaK also works in tandem with var- ious other chaperones. For example, the DnaK is known MATERIALS AND METHODS to collaborate with HtpG (E. coli Hsp90) for client pro- tein remodeling [19] and ClpB during protein disaggre- Expression and Purification of DnaK, DnaJ, and gation [20{22]. Importantly, DnaK serves as the central GrpE organizer in E. coli chaperone network to control pro- teostasis [23]. DnaK, DnaJ, and GrpE proteins were obtained as fol- While many aspects of DnaK have been elucidated, lows. The dnak, dnaj, and grpe genes were each cloned questions remain on how DnaK binding to protein sub- into the pNIC28-Bsa4 vector [33]. BL21(DE3) Rosetta strate may facilitate protein folding. Previously, DnaK competent cells were transformed with each of the recom- was assumed to act as a holdase or an unfoldase [24, 25]. binant plasmids. The cells were cultivated in TB media A holdase refers a chaperone binding to an unfolded supplemented with 100 µg/mL kanamycin and 34 µg/mL substrate that decreases the rate of transition to the chloramphenicol in a LEX system (Harbinger Biotech). folded state. By binding to exposed hydrophobic re- Overexpression of proteins was induced with 0.5 mM gion of unfolded or disaggregated protein segments, a IPTG overnight at 18 ◦C. The cells were then harvested holdase is expected to slow down the protein folding to by centrifugation and resuspended in lysis buffer (100 prevent premature folding that may result in misfold- mM HEPES, 500 mM NaCl, 10 mM Imidazole, 10 % ing and/or aggregation. In contrast, an unfoldase refers glycerol, 0.5 mM TCEP, pH 8.0) containing 1000 U ben- to a chaperone that increases the unfolding rate of a zonase (Merck) and 1000x dilution protease inhibitor folded/misfolded/aggregated substrate to facilitate the cocktail (Calbiochem). Following this, the cells were son- proteins to refold to their native conformation. The ex- icated and the lysates were centrifuged at 47,000 g, 4 ◦C. tent to which DnaK employ these mechanisms warrants for 25 min. The supernatants were filtered through 1.2 further investigation. µm syringe filters and loaded onto IMAC columns in an In this article, we aim to address the above questions AKTA Xpress system (GE Healthcare). Bound proteins by using single-molecule protein refolding assay, which were washed with buffers containing up to 25 mM imida- can provide a direct insight into how chaperone activity zole and finally eluted with buffer containing 500 mM im- interferes directly with protein refolding. This method is idazole. Eluted proteins were loaded onto HiLoad 16/60 based on magnetic tweezers that is capable of applying Superdex 200 columns (GE Healthcare) equilibrated with force to a protein domain and modulating the balance gel filtration buffer (20 mM HEPES pH 7.5, 300 mM between the folded and the unfolded states [26].
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