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Refolding Activity of Bacterial Hsp90 in Vivo Reveals Ancient Chaperoning Function

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Refolding Activity of Bacterial Hsp90 in Vivo Reveals Ancient Chaperoning Function bioRxiv preprint doi: https://doi.org/10.1101/462549; this version posted November 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Refolding activity of bacterial Hsp90 in vivo reveals ancient chaperoning function Tania Morán Luengo1,2, Toveann Ahlnäs1,2,3, Anna T. Hoekstra2,4, Celia R. Berkers2,4, Matthias P. Mayer5, Stefan G. D. Rüdiger1,2* 1 Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands 2 Science for Life, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands 3 Åbo Akademi University, Tuomiokirkontori 3, 20500 Turku, Finland 4 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands 5 Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH-Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany * Corresponding author: SGDR ([email protected]) ABSTRACT of Hsp70, which together constitute an effective folding cascade [6]. This cascade acts early on the folding path, The conserved molecular chaperones Hsp70 and preparing their client for subsequent folding by itself. Hsp90 play a key role in protein folding. Mechanistically, Hsp90 acts downstream from A key step in this process is the transfer of the client from Hsp70 solving an Hsp70-inflicted folding block. It is Hsp70 to Hsp90. This step stringently depends on the unclear, though, when and to which extend the ATPase activity of Hsp90. [6-8]. Competitive inhibitors for concerted action of this cascade becomes crucial in ATP binding such as Geldanamycin and Radicicol act as living organisms. Here we show that, in E. coli cells, potent conformational and functional Hsp90-specific Hsp90 dramatically improves protein refolding after blockers [9-12]. In eukaryotes, Hsp90 is involved in heat stress while it is dispensable for de novo maturation and assembly of a wide array of protein folding. We found that Hsp90 inhibition effectively substrates, many of which are involved in signalling reduced the refolding yields in vivo, leading to processes [13, 14]. Recently, we have shown that Hsp90 strongly reduced enzymatic activity of the also has a conserved key function in protein folding, paradigmatic chaperone client luciferase and solving an Hsp70-inflicted folding impasse [6]. Effective broadly increased aggregation of the E. coli protein folding is crucial for all organisms, including proteome. Additionally, the presence of Hsp90 bacteria. The bacterial Hsp90 system, HtpG, thus, offers the during refolding reduces the net ATP consumption possibility to study when and under which circumstances presumably by sparing the substrate binding-and- release cycles on Hsp70. This mechanism explains the Hsp90 function in folding is crucial for the cell. how the cooperation of Hsp90 with the Hsp70 chaperone system creates robust folding machinery Here we show that E. coli Hsp90 plays a fundamental role in a sustainable manner. Together, we describe a in protein refolding after heat-shock in the cellular general function for bacterial Hsp90 as a key factor environment. Using the paradigmatic chaperone substrate of the folding cascade, which may be the ancient firefly luciferase, we found that in vivo Hsp90 plays a key activity of this evolutionary conserved machine. role in refolding after stress, while it is not essential for effective folding of nascent luciferase. The refolding Keywords: Molecular chaperones / E. coli / Hsp90 / Hsp70 / function of Hsp90 is key to increase solubility of the global Protein Folding / Protein quality control pool of cellular substrates after stress depending unfolding. The effectivity of the Hsp70-Hsp90 cascade also becomes INTRODUCTION evident when monitoring the ATP consumption, which we found to be 3-fold lower than for Hsp70 alone. Molecular chaperones assist protein folding in the cell [1, 2]. The coordinated action of the conserved ATP- dependent Hsp70 and Hsp90 chaperone machines plays a RESULTS AND DISCUSSION key role for the attainment of the folded and mature condition of many proteins [3-6]. Hsp90 acts downstream Hsp90 is dispensable for de novo folding Morán Luengo et al. 1 Ancient function of Hsp90 in vivo | BioRxiv bioRxiv preprint doi: https://doi.org/10.1101/462549; this version posted November 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. multidomain protein, heterologous for E. coli and presents low refolding efficiency in the absence of chaperones, which makes it representative of many other substrates allowing generalisation [15, 16]. We confirmed that, in our hands, luciferase refolds in vivo after heat-induced denaturation in E. coli cells. We used an IPTG-inducible expression system to recombinantly produce luciferase in E. coli by induction before the stationary phase. We halted total translation by inhibiting the ribosome with tetracycline before temperature-induced unfolding of luciferase, ensuring the presence of a homogeneously unfolded luciferase population (Fig. 1A). After a 20-minute heat-shock at 42ºC, when luciferase reached ~ 98% unfolding, we monitored the protein refolding over time. Luciferase produced in E. coli cells, containing both Hsp70 and Hsp90 wild type proteins, recovered up to ~60% after 45 min upon temperature downshift (Fig. 1B). This sets the scene for assaying the effect of Hsp90 in de novo folding and after thermal stress. First, we tested the in vivo role of the bacterial Hsp90 for de novo folding. As Hsp90 function is tightly coupled to its ATPase activity, we used the Hsp90-specific ATPase inhibitors Geldanamycin and Radicicol to investigate the effect of Hsp90 in luciferase folding [10, 17, 18]. To assess the impact of Hsp90 inhibition in de novo protein folding, we blocked Hsp90 ATPase activity in E. coli cells by increasing concentrations of Geldanamycin and Radicicol (0 – 60 µM) before production of luciferase allowing to assess the impact of Hsp90 inhibition in de novo protein folding (Fig. 1C). The amount of active luciferase did not Figure 1 - Hsp90 has only a mild influence in de novo decrease upon titration of each of the inhibitors (Fig. 1D). protein folding. We conclude that Hsp90 is not essential for de novo folding A) Experimental set-up for the luciferase refolding after of luciferase. This is remarkable as it is a paradigmatic heat-shock in E. coli shown in B). B) In vivo recovery of the luciferase activity after 20 minutes heat-induced chaperone substrate. denaturation. Luciferase recovery over time is plotted against the recovery time (±SEM n = 3). C) Experimental set up for the effect of Hsp90 inhibition in de novo folding. Hsp90 plays a key role in vivo in refolding after D) Geldanamycin and Radicicol have a weak effect in heat stress decreasing the production of active luciferase. Luciferase activity after production is plotted against increasing The function of Hsp90 is mechanistically important in concentrations of the inhibitors (±SEM n = 3). protein refolding downstream from Hsp70 [6]. However, protein refolding and de novo folding strikingly differ in the To study the effect of Hsp90 in folding, we monitored availability of the folding domains, the protected bioluminescence of the model substrate luciferase from environment created by the ribosome and the chaperones Photinus pyralis in living E. coli cells. This approach takes involved in the process [19]. In bacteria, newly synthesized advantage of the fact that only fully folded luciferase is able proteins are welcomed by the ribosome-associated to perform its enzymatic reaction. The reaction requires the chaperone Trigger Factor (TF) [22]. TF and the Hsp70 firefly substrate D-luciferin, which is able to enter bacterial homologue, DnaK, have combined activity and unpermeabilized E. coli cells and produces light that can be overlapping binding specificities [20, 21]. This implies that measured in vivo [15]. Luciferase is a monomeric Morán Luengo et al. 2 Ancient function of Hsp90 in vivo | BioRxiv bioRxiv preprint doi: https://doi.org/10.1101/462549; this version posted November 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. in the presence of TF, DnaK might not have access to the expressed luciferase in E. coli cells, and subsequently nascent chain and thus Hsp90 action is not required. treated them with Geldanamycin or Radicicol (Fig. 2A). Instead, upon stress conditions a big number of proteins are After 10 min incubation in the presence of the inhibitor, the suddenly unfolded and prone to aggregate. In those cells were subjected to heat stress for 20 min at 42ºC in situations, Hsp70 is essential for aggregation prevention order to achieve complete substrate unfolding, and we and subsequent refolding [22]. To investigate the function monitored the refolding of luciferase at different time of Hsp90 in this process downstream from Hsp70, we points. Figure 2. Hsp90 plays a key role in substrate activity
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