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ScienceDirect Non-equilibrium conformational dynamics in the function of molecular chaperones Alessandro Barducci and Paolo De Los Rios Why do chaperones need ATP hydrolysis to help proteins reach their native, functional states? In this review, we highlight the most recent experimental and theoretical evidences suggesting that ATP hydrolysis allows molecular chaperones to escape the bounds imposed by equilibrium thermodynamics. We argue here that energy consumption must be fully taken into account to understand the mechanism of these intrinsically non-equilibrium machines and we propose a novel perspective in the way the relation between function and ATP hydrolysis is viewed. Address Laboratoire de Biophysique Statistique, School of Basic Sciences, E´cole Polytechnique Fe´de´rale de Lausanne (EPFL), CH 1015 Lausanne, Switzerland Corresponding authors: Barducci, Alessandro ([email protected]) and De Los Rios, Paolo ([email protected])

Current Opinion in Structural Biology 2015, 30:161–169 This review comes from a themed issue on Folding and binding

aggregation [4–6]. Several chaperone families have been identified to date, such as Hsp60, Hsp70, Hsp90, Hsp100 [7,8,9]. The number in their name indicates their approximate molecular mass, in kiloDaltons (kDa), and the acronym Hsp stands for Heat shock protein, because their experimental correlation with thermal stress has provided the first evidence for their existence, and suggested their cellular role. The different Hsp families share some general features: they are ubiquitous across most domains of life and have a high degree of sequence conservation, hinting that they are an ancient and efficient solution for the safeguard of protein homeostasis. The crucial role of chaperones is further highlighted by the observation that all organisms stringently rely on their assistance for survival under a disparate array of stresses. Moreover, chaperones (prominently Hsp70 and Hsp90) have also taken up a host of constitutive, housekeeping and regulatory roles under non-stressful conditions [10,11].

Edited by Annalisa Pastore and Guanghong Wei For a complete overview see the Issue and the Editorial Available online 13th March 2015 http://dx.doi.org/10.1016/j.sbi.2015.02.008 0959-440X/# 2015 Elsevier Ltd. All rights reserved.

Introduction The physical and chemical properties of protein sequences determine their native states [1,2] within a physiological range of environmental conditions, but might not be able to guide the folding process in the crowded environment of the cell and under stresses, such as abrupt temperature increases. Proteins that are too slow at finding their native structure are at risk of residing into unfolded or misfolded conformations that are non-functional and are prone to further inappropriate interactions, leading to potentially cytotoxic aggregates. Because the individual amounts of all proteins in the cell, as well as their solubility, are likely fine-tuned within a limited range for optimal cellular function, any perturbation of this protein homeostasis can have far-reaching consequences [3]. Molecular chaperones are a class of proteins that protect cells against the noxious effects of protein misfolding and www.sciencedirect.com

Despite the great relevance of chaperones for the inner workings of cells, their precise molecular mechanism of action has not been unambiguously elucidated. In vitro, chaperones reduce aggregation and promote native folding possibly by two different but not mutually exclusive mechanisms, namely holding and unfolding. On the one hand, Hsps can bind (hold) unfolded and/or misfolded polypeptides before they have a chance to aggregate, thus decreasing their concentration and the associated aggregation rate. As a consequence, aggregation is tamed and native refolding becomes the dominant process. On the other hand chaperones can also rescue stable pre-aggregated and misfolded proteins, a process that requires some form of remodeling (unfolding) of the bound substrates [8,12–14]. Hsps are also characterized by the remarkable variety of conformations that they can access, which has been progressively unveiled in recent years and that has been recognized as crucial for their function [15].

The function of molecular chaperones stringently depends on ATP hydrolysis There is ample evidence that ATP and the ATPase activity are stringently required for the function of heat-shock proteins. Several ATPase defective mutations of the Escherichia coli Hsp60 chaperonin, GroEL, were not able, in vivo, to rescue GroEL deficient strains [16,17]. The same mutants have been shown to be also defective Current Opinion in Structural Biology 2015, 30:161–169

162 Folding and binding

in vitro [17]. Likewise, non-hydrolyzing mutants of DnaK, the E. coli Hsp70 ortholog, were not able to complement the temperature sensitivity phenotype of a DdnaK strain and to promote proliferation of bacteriophage l in vivo [18–20], and to allow the replication of l DNA and the refolding of firefly luciferase in vitro [20,21]. Saccharomyces cerevisiae Hsp90 depends on its ATPase activity for in vivo function, as mutations impairing ATP hydrolysis are incompatible with cell growth [22]. Arabidopsis thaliana cells where Hsp100 chaperones carry a mutation compromising nucleotide hydrolysis activity do not acquire tolerance to temperature stress [23].

Non-equilibrium versus equilibrium conformational cycles Our current understanding of proteins has evolved from the initial static picture provided by the first X-ray structures and it recognizes that they are highly dynamical molecules, which can sample several diverse conformational states [24,25]. This conformational dynamics is often tightly coupled with binding of other biomolecules, such as substrates and other co-factors, and it defines the states and the overall free-energy landscape of the system (Figure 1). At thermodynamic equilibrium, the occupation of each state is dictated by its free energy and the transitions between them are driven by thermal fluctuations according to rates that are determined by the freeenergy barriers. In this scenario the system evolution is governed by the detailed balance rule, so that no net fluxes are present on the landscape (Figure 1a). According to a widespread view of ATPase chaperones, the main role of ATP hydrolysis is to change the nature of the bound nucleotide, thus selecting disparate sets of conformations (different minima in the landscape, cf. Figure 1a) with their advantageous features, such as different affinities for client proteins.

This rather static description, whereby ATP hydrolysis and nucleotide exchange simply choose the conformations whose equilibrium properties are more convenient, is challenged by a comprehensive analysis of non-equilibrium biochemical cycles driven by energy consumption. Indeed, the ATP and ADP concentrations in the cell are kept far from their equilibrium values, with ATP about tenfold more abundant than ADP, due to the energy consuming action of ATP-synthases. As a consequence, proteins such as chaperones, whose conformational dynamics is coupled to ATP hydrolysis, are characterized by non-equilibrium conformational cycles. Particularly, as the rate of ATP synthesis by chaperones is negligible [26], the backward conversion from the ADPbound state to the ATP-bound state has to proceed through nucleotide exchange, which does not obviously correspond to the reverse process of hydrolysis. This simple observation has profound implications, because the populations of the ATP-bound and ADP-bound states are not anymore thermodynamically determined by their free-energy differences, but are instead kinetically controlled by the ATPase and nucleotide exchange rates (Figure 1b). Furthermore, the presence of unbalanced hydrolysis-driven fluxes between some pairs of states (red arrow, Figure 1b) induces fluxes over the landscape (green arrows, Figure 1b), owing to the tendency of the system to flow back toward equilibrium. The presence of currents over cycles can be measured, allowing the quantification of the displacement of the system from equilibrium conditions. Indeed, at equilibrium the clockwise and anticlockwise fluxes over a cycle are equal, whereas their ratio in non-equilibrium is related to the amount of consumed energy [27]. Intuitively, these cyclic currents are necessary to perform any kind of work, whereas no work can be extracted by the simple thermal fluctuations characteristic of thermodynamic equilibrium.

Figure 1

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The free-energy landscape in equilibrium and non-equilibrium conditions. Schematic drawing of a free-energy landscape, here represented as a function of two coordinates. Several minima, each representing an ensemble of states, are present and are occupied according to their equilibrium probabilities (a), that are here captured by filling the basins all at the same level. At equilibrium the exchange fluxes between states cancel pairwise, and no net fluxes are present. (b) In non-equilibrium conditions irreversible processes (e.g. ATP hydrolysis) drive some of the transitions. In this case the net flux (red arrow) is not pairwise compensated and the system relaxes along different pathways (green arrows). A new steadystate establishes, where the stationary probabilities do not correspond to the equilibrium ones (in panel b blue rings mark the equilibrium level of panel a, to highlight the difference from the non-equilibrium scenario). Current Opinion in Structural Biology 2015, 30:161–169

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Molecular chaperones as non-equilibrium machines Barducci and De Los Rios 163

Hsp70 chaperones and ultra-affinity Hsp70s are possibly the simplest and best characterized ATP-dependent molecular chaperones and provide a clear example of the functional coupling of ATP hydrolysis and substrate interaction in a non-equilibrium cycle. Hsp70s are ubiquitous and highly conserved proteins, whose structure comprises a N-terminal ATPase Nucleotide Binding Domain (NBD, red in Figure 2a) and a C-terminal Substrate Binding Domain (SBD, purple and cyan in Figure 2a) [28]. Binding of ATP prompts the chaperone in a conformation with fast substrate binding and unbinding rates, while ADP-bound Hsp70 is characterized by much slower (two to three orders of magnitude) substrate exchange rates [29–31]. The conversion of one conformer to the other can take place either by nucleotide exchange (an equilibrium process driven by thermal fluctuations, blue vertical arrows in Figure 3a) or by ATP hydrolysis (a non-equilibrium process, red vertical arrows in Figure 3a). The basal hydrolysis rate of Hsp70 chaperones is typically slow (10 4 to 10 3 s 1), but is greatly enhanced, up to 1 s 1, by the concomitant presence of substrates and accompanying ATPase stimulating cochaperones, that is, the J-domain containing proteins (JDPs) [32].

Remarkably, according to fluorescence experiments on peptides [31,33], the affinities of the ATP-bound and ADP-bound states for substrates do not differ much, their ratio spanning at most one order of magnitude, typically in favor of the latter. If Hsp70s did not consume energy, for any chaperone solution composed of two subpopulations of ATP-bound and ADP-bound conformers the laws of equilibrium thermodynamics would apply. As a consequence, the overall affinity for the substrate would be inescapably constrained to be a weighted average of the affinities of the two nucleotide-bound states, thus never exceeding the ADP-bound state affinity [34] (Figure 3b, green-shaded region). This is not what has been observed in experiments where, in the presence of ATP and JDPs, Hsp70s bind their substrates more efficaciously than in the presence of ADP, which would naively induce the highest affinity [35,36]. Instead, the constant hydrolysisdriven cycling of Hsp70s through the ATP-bound and ADP-bound states offers continuous chances of fast substrate binding to the ATP-bound conformation (fast substrate-exchange kinetics), while unbinding takes place either from the ADP-bound state (slow substrate-exchange kinetics) or from the ATP-bound state after at least one substrate-stimulated ATPase cycle, as originally hinted by Misselwitz et al. [35] and Zuiderweg et al. [37].

Figure 2

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The structures of Hsp70, Hsp90 and Hsp60-GroEL. (a) Hsp70 chaperones are composed of a Nucleotide Binding Domain (NBD, red), where ATP binds and is hydrolyzed, and of a Substrate Binding Domain (SBD), which is in turn made of a b-basket (purple) and of a helical lid (cyan). In the ATP-bound state (represented here) the b-basket the helical lid are docked on the NBD, whereas they undock from it in the ADP-bound state and close with each other as in a clamp. (b) Hsp90 chaperones are homodimers (here one protomer is portrayed in ribbon representation and the other in translucant space-filling form). Each subunit is composed by N-terminal (cyan), middle (darker green) and C-terminal (lighter green) domains. Dimerization takes place through the C-terminal domain, although in the ATP-bound state (represented here) the two protomers interact also through the N-terminal domains. In the ADP-bound state, Hsp90s are highly dynamic and can take both closed and open conformations. (c) Hsp60-GroEL chaperonins are composed by two back-to-back stacked rings, each made of seven identical protomers (here one protomer is portrayed in ribbon representation and the others in translucant space-filling form). Each protomer comprises an apical domain (red), a middle domain (orange) and an equatorial domain (yellow). Interactions between the two rings take place through the equatorial domains, whereas interactions with substrates and with GroES co-chaperones (Hsp10, here represented in brownish translucent space-filling representation) take place through the apical domains in the ATP-bound ring. www.sciencedirect.com

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164 Folding and binding

Figure 3

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The Hsp70 non-equilibrium cycle and ultra-affinity. (a) The biochemical cycle of Hsp70 chaperones is coupled to substrate binding/unbinding. Hsp70s can assume two conformations: with an open substrate-binding region, stabilized by ATP, and with a closed substrate-binding region, stabilized by ADP. The transitions from one conformation to the other can take place by thermally driven nucleotide exchange processes or by ATP-hydrolysis (red arrows). Substrate binding in the ATP-bound state accelerates ATP hydrolysis with respect to the substrate-free ATP-bound chaperone (khS > > k h ), with consequent clamping of the chaperone on the substrate. The thermodynamic dissociation constant of the substratechaperone complex is lower by one order of magnitude in the ADP-bound state. Substrate access to and egress from the ATP-bound conformation are orders of magnitude faster than the same processes to and from the ADP-bound state. (b) Effective dissociation constant of substrate-Hsp70 complexes as a function of substrate-induced hydrolysis acceleration, khS =k h . The green-shaded region corresponds to the range of values that would be accessible at equilibrium (no hydrolysis), comprised between the substrate dissociation constant of the ATP-bound and ADP-bound states. The red/yellow region (ultra-affinity region) is reachable only by energy consumption, and the effective dissociation constant can be three orders of magnitude smaller than the one that, at equilibrium, would be accessible with the cellular ATP and ADP concentrations. This figure has been adapted from [34].

The ensuing effective substrate dissociation rate can be several orders of magnitude smaller than the equilibrium one, resulting in a correspondingly smaller non-equilibrium dissociation constant (nM instead of mM, red-yellow region in Figure 3b). This non-equilibrium ultra-affinity [34] rationalizes experimental observations, and might have far-reaching functional implications. Indeed, during Hsp70-driven import of proteins into mitochondria Current Opinion in Structural Biology 2015, 30:161–169

and endoplasmic reticulum, the enhanced affinity could ensure strong binding also to sequences that are not, per se, good binders [35], so to ensure a reliable grip to apply the entropic pulling force [38]. Similarly, Hsp70s might exploit ultra-affinity to bind to almost any sequence emerging from the ribosomal tunnel to alleviate ribosome stalling by entropic pulling, as has been recently proposed [39]. www.sciencedirect.com

Molecular chaperones as non-equilibrium machines Barducci and De Los Rios 165

As guardians of protein homeostasis, Hsp70s need to be able to find and neutralize misfolded and unfolded polypeptides even at low concentrations before they turn into cytotoxic aggregates; at the same time, Hsp70s must not be specific, because any protein can in principle need their assistance. High affinity and promiscuity are difficult to achieve together at equilibrium, as the former is typically attained by fine-tuned complementarity between the specific binding partners. This difficulty can be circumvented in a non-equilibrium scenario where effective affinities are kinetically determined and not directly related to equilibrium free energy differences. Several lines of evidence support the view of Hsp70s as unfolding machines. Stable misfolded luciferase monomers were reactivated by Hsp70s, together with their co-chaperones and ATP, through a process that involved the significant unfolding of the non-native substrate upon chaperone binding, followed by native refolding after substrate release [40,41]. Kellner et al. [42] have shown by means of single-molecule experiments and simulations that Hsp70 binding leads to an expansion of unfolded rhodanese. Strikingly, the substrate expansion was abolished by the progressive conversion of ATP into ADP by hydrolysis, suggesting that the affinity of ADP-bound Hsp70s for their substrates, although the largest, was not sufficient to ensure strong binding and consequent substrate expansion. Likewise, the regulatory unfolding action of Hsp70s on the glucocorticoid receptor stringently depends on ATP and ATP hydrolysis, because ADP alone is not able to induce the necessary Hsp70 binding and the concomitant loss of structure leading to the inactivation of the receptor [43]. Ultra-affinity would thus be crucial for both the holding and the unfolding functions of Hsp70 chaperones.

Multiple opportunities for non-equilibrium control in the dynamic cycle of Hsp90 chaperones Hsp90s are highly conserved chaperones present in prokaryotic and eukaryotic organisms, and are among the most abundant proteins in the cell [44–46]. They are composed of three domains, and the functional form is a homodimer of two protomers interacting through their N-terminal and/or C-terminal domains (Figure 2b) [7,11]. Interaction with the substrate can take place on one or more Hsp90 domains [47–51], but the action of Hsp90 chaperones on their substrates has not been unambiguously characterized. Despite this lack of information, Hsp90s have recently taken center stage in molecular biology because the key oncogenic suppressor p53 is one of the Hsp90 substrates [52–54], together with a plethora of other kinases [55]. Furthermore, there are encouraging reports about the clinical efficiency of Hsp90 inhibitors in cancer therapy [56]. The Hsp90 homodimer can access a large number of conformations because of the relatively flexible linkers www.sciencedirect.com

connecting the three domains of each protomer [46]. Recently, single-molecule experiments have investigated the coupling between this conformational plasticity and the nature of the bound nucleotide. The conformational fluctuations of prokaryotic Hsp90 (HtpG) were found to depend on ATP or ADP binding, and could thus be directly modulated by ATP hydrolysis [57]. In particular, according to Ratzke et al. [57] binding of ATP would stabilize the closed conformation (characterized by contacts between the two N-terminal domains and between the two C-terminal domains), which is more favorable for irreversible hydrolysis. ATP binding would thus act as a ratchet that provides directionality to the biochemical cycle of HtpG. Eukaryotic Hsp90 showed instead nucleotide-independent thermal fluctuations between open and closed conformations [58,59], that were brought again under the control of ATP by co-chaperones (that are purportedly absent in bacteria). In eukaryotes, thus, ATP and co-chaperones are both needed to restore the ratchet mechanism, and thus directionality [60]. Notably, in the presence of p23, it has been found that the cycle flux in the direction of hydrolysis exceeds the flux in the opposite direction, a clear indication of the nonequilibrium nature of the Hsp90 cycle [60]. The pronounced conformational flexibility of Hsp90s is likely to play a crucial role for their interactions with client proteins. Indeed, conformational flexibility suggests that the free-energy differences and barriers between the various conformers are small, and thus the equilibrium should be affected by the very binding of the substrates. Because each conformation is characterized by different hydrolysis and nucleotide exchange rates, substrates binding and unbinding could thus easily modulate the Hsp90 biochemical cycle [50,61]. All these features are likely to result into remarkable yet uncharacterized properties of the Hsp90 cycle. Indeed, even if the observation that substrate release is enhanced after ATP hydrolysis [11] is not compatible with a straightforward extension of the ultra-affinity concept to this chaperone, other features brought about by nonequilibrium, akin to what observed in Hsp70, might characterize its cellular functioning. Although in eukaryotes a detailed analysis may be hindered by the large number of regulating co-chaperones [11], prokaryotic Hsp90 might represent an excellent opportunity to investigate the coupling between hydrolysis and interactions with substrates.

The evidence for non-equilibrium effects in the biochemical cycle of Hsp60 chaperonins Chaperonins are allosteric complexes formed by two back-to-back, stacked rings of protomers each encircling a cavity (Figure 2c). In E. coli chaperonins (GroEL) each ring is a heptamer made of identical Hsp60 monomers [62]. Each protomer is composed of apical, middle and Current Opinion in Structural Biology 2015, 30:161–169

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equatorial domains. The apical domains line the entrance of the internal cavity of the ring, while the equatorial domains provide the surface of interaction between the two rings. Although chaperonins are perhaps the most studied of the ATPase chaperones, their precise mechanism of action has not been completely elucidated. Unfolded or misfolded proteins are known to bind to the apical domains of the apo-heptameric (cis-)ring, where they might undergo some form of unfolding [63,64]. Upon ATP binding, the apical domains change orientation prompting the release of the substrate, and the further binding of GroES (Hsp10) co-chaperones, that seal the cavity. There, trapped substrate could refold in a protected environment that shelters them from aggregation. Recent results have also suggested that the cavity itself might accelerate folding, because of the properties of its surface [65]. ATP hydrolysis in the cis-ring and ATP binding in the trans-ring lead then to GroES dissociation and release of the substrate that, if still unfolded or misfolded, could undergo a new binding and release cycle. According to the present understanding, thus, ATP hydrolysis and ATP binding would act as timers to set the duration of the cycle. Furthermore, the ATP cycle would be strictly coupled to some form of negative cooperativity between the two rings, whereby each nucleotide binding and hydrolysis turnover in one ring would prime the opposite ring to start a similar cycle, with a 1808 phase difference. The full set of ATP-bound and ADP-bound conformations has purportedly been recently resolved, providing a structural-level description of the GroEL/S-substrate nucleotide-dependent interactions [66,67]. Although the multiplicity of the nucleotide-dependent conformations accessible to GroEL, each with its own substrate affinity, is being progressively unveiled, their inter-conversion rates crucially determine the function of Hsp60s. Indeed, recent single-molecule experiments on the single-ring Hsp60 mutant SR1 have highlighted that the progression of the chaperone through its ATPase cycle, paced by the rates of ATP binding, ATP hydrolysis and ADP release, results in a non-equilibrium conformational ensemble (cf. Figure 1b) [68], where the concentrations of molecules in each conformation is crucially dictated by the hydrolysis rate. This suggests that moving away from equilibrium conditions allows controlling, by way of ATP hydrolysis, how different states are occupied. As a matter of fact, all measured rates in the cycle likely lie in a limited range spanning one to two orders of magnitude. Ye and Lorimer [69] have also shown that the presence of a bound substrate protein can increase the rate of nucleotide exchange up to two orders of magnitude, thus coupling the system to the intrinsic time-scale of the unfolded-to-native transition of the substrate. These results would suggest that some transitions, whose basal rate is very slow, might be accelerated by the Current Opinion in Structural Biology 2015, 30:161–169

interaction with the substrate, analogously to what observed for the hydrolysis rate of Hsp70. In turn, only future experimental and theoretical studies will shed light on the details of the cycle of Hsp60s, paving the way for a comprehensive picture of non-equilibrium effects on the chaperonin function.

Other chaperones This necessarily short overview of the relation between the function of molecular chaperones and energy consumption in the form of ATP hydrolysis has left aside small Heat shock proteins (sHsp) that do not bind ATP and are thus traditionally considered non-ATPase chaperones [70]. SHsps are small (approximately 12–43 kDa) proteins characterized by the presence of a a-crystallin domain [71]. They are believed to passively bind misfolded and unfolded proteins, thus sequestering them and slowing down their aggregation, in agreement with the chaperone holding mechanism of function [71]. SHsps are found in solution both as monomers or small oligomers, that bind substrates, and as ordered supra-molecular complexes containing up to tens of protomers. Very recently it has been observed that sHsp phosphorylation can control their degree of oligomerization [72], and in turn their function [73]. Because each phosphorylation and dephosphorylation cycle consumes an ATP molecule, and therefore energy, it might be due time to consider sHsps as non-equilibrium, ATP-driven machines. As a matter of fact, phosphorylation/dephosphorylation cycles, regulated by specific kinases and phosphatases, are used by cells to make signaling molecules ultrasensitive to environmental cues [74]. Ultrasensitivity is associated with steep sigmoidal, rather than simpler linear or Michaelis–Menten-like, response curves, which could not be achieved without energy consumption. It can therefore be expected that ATP-derived covalent modifications could also bestow hitherto unknown properties on sHsps.

Conclusions It is well established that most chaperones need ATP to assist protein homeostasis, and in several cases the structural modulations induced by ATP and ADP binding have been characterized while their consequences on chaperone/substrate interactions are less extensively understood. All these information are necessary but not sufficient to achieve a molecular description of the mechanisms of function of chaperones. Indeed, energy consumption due to ATP hydrolysis moves the chaperone conformational dynamics away from thermodynamic equilibrium, so that the chaperone cycle results into a non-equilibrium steady-state, which is non-trivially governed by all the kinetic rates of the system. www.sciencedirect.com

Molecular chaperones as non-equilibrium machines Barducci and De Los Rios 167

As exemplified by the case of ultra-affinity of Hsp70s, the mixing of time-scales that is a consequence of the kinetic, rather than thermodynamic control of the cycle, can endow chaperones with advantageous properties that are crucial for their functions. Only recently a few studies have started to shed light on non-equilibrium features in chaperone functioning and, as a consequence, this review is biased to some extent toward these results. We hope that the ideas and concepts that we have highlighted here will stimulate a novel way to consider the function of chaperones, and as a consequence new experiments and theoretical models. Indeed, only a true integration of structural, biochemical, single-molecule and theoretical approaches has the potential to bring us closer to a definitive picture of molecular chaperones. From a broader point of view, we should always keep in mind that evolution has randomly tinkered with the laws of physics, preserving and constantly tuning solutions that are useful while discarding, along the way, what was underperforming. Non-equilibrium thermodynamics has always been there to be exploited by living systems. As a consequence, its full inclusion among the design principles that are used to describe cellular processes is mandatory if we want to grasp the inner workings of the cell.

Conflict of interest statement We have no conflicts of interest.

Acknowledgement AB thanks the Swiss National Science Foundation for financial support under the Grant PZ00P2_136856.

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