Cells cope with excessive heat, toxic compounds and other adverse environmental conditions by triggering an internal repair process called the heat shock response. In mammalian cells, a protein called Hsf1 is activated by stress and regulates the activity of a large set of target genes. These genes code for proteins that help the cell cope with the effects of stress, for example, by repairing or breaking down damaged proteins. Under normal conditions, Hsf1 exists as a single molecule, but when it is activated, three molecules come together to make a complex called a trimer that is able to bind to DNA and activate the target genes.

Proteins are made of long chains that then fold into specific three-dimensional shapes. It is not known how Hsf1 is kept in an inactive state in healthy, unstressed cells. One possibility is that the protein folds into a three-dimensional shape that prevents it from being activated. Alternatively, Hsf1 may be bound to other proteins called chaperones that move away when the cell is under stress because they are needed to help the damaged proteins refold into their own three-dimensional shapes.

Hentze et al. used a variety of biochemical techniques to study the human Hsf1 protein. The experiments showed that there are two regions of the Hsf1 protein that changed shape dramatically when the temperature increased. A region that regulates the activity of Hsf1 unfolded, while a region involved in making the trimer became more stable. Detailed analysis showed that once the regulatory region unfolded, the protein was able to interact with other Hsf1 units to make the trimer. Therefore, Hsf1 can directly sense and respond to changes in temperature without the aid of any chaperone proteins.

Further experiments showed that the formation of Hsf1 trimers and the ability of these trimers to bind to DNA depend upon both the temperature and the amount of Hsf1 present. In addition, a chaperone protein called Hsp90 – which is known to be able to interact with Hsf1 – influenced how Hsf1 responded to changes in temperature. Hentze et al. also present a model for the activation of Hsf1 that allows for flexibility in the response of Hsf1 to changes in temperature. Previous studies have shown that Hsf1 is chemically modified during stress and also while the cell recovers from stressful conditions. Therefore, the next challenge will be to find out how these modifications influence the way in which Hsf1 responds to stress.

To cope with changes in physical and chemical properties of the environment as well as with physiological and pathophysiological conditions which cause protein misfolding, organisms mount a homeostatic transcriptional program, the so-called heat shock response (Jolly and Morimoto, 2000). In all eukaryotic cells, heat shock transcription factor (HSF) 1 is the master regulator of this response and alters transcription of a large number of genes, some of which encode chaperones and proteases (Anckar and Sistonen, 2011). Although this response is essentially cell autonomous, systemic modulation of this response has been observed in metazoa (Morimoto, 2008; Prahlad et al., 2008; Prahlad and Morimoto, 2011).

Metazoan Hsf1 consists of a N-terminal winged helix-turn-helix DNA binding domain (Harrison et al., 1994; Vuister et al., 1994), a hydrophobic shorter heptad repeat regions (HR-A/B) proposed to function as a leucine zipper coiled-coil trimerization domain (Clos et al., 1990; Rabindran et al., 1993), a regulatory domain, a second heptad repeat (HR-C) and a C-terminal transcription activation domain (Figure 1A) (Anckar and Sistonen, 2011; Voellmy, 2004). In unstressed cells metazoan Hsf1 is monomeric and supposed to be in complex with molecular chaperones, including Hsp70, Hsp90 and TRiC/CCT (Shi et al., 1998; Zou et al., 1998; Neef et al., 2014). At physiological concentrations monomeric Hsf1 does not bind appreciably to heat shock elements (nGAAn). In the activated state Hsf1 forms trimers or higher order oligomers and binds to its response elements in heat shock gene promoters (Clos et al., 1990; Rabindran et al., 1993). Currently, two models are discussed for the heat-induced activation of Hsf1: (1) Based on the observation that deletion or mutational alteration of HR-C leads to constitutive trimerization Wu and co-workers proposed that Hsf1 is a thermosensor itself and kept monomeric by intramolecular leucine zipper formation (Rabindran et al., 1993). However, activation of human Hsf1 in human and insect cells and Xenopus oocytes occurs at different temperatures, arguing against an Hsf1 intrinsic mechanism of heat shock activation (Baler et al., 1993; Clos et al., 1993). (2) Owing to the fact that the large variety of Hsf1-inducing signals have in common to cause protein misfolding and in analogy to the regulation of the heat shock response in E. coli (Guisbert et al., 2008), chaperones were proposed to prevent Hsf1 activation and to be titrated away from Hsf1 under stress conditions, resulting in heat shock response induction (Morimoto, 1998). Consistent with this hypothesis is the observation that inhibition of Hsp70, Hsp90 or TRiC/CCT or knock-down of their expression leads to the induction of the heat shock response (Powers and Workman, 2007; Powers et al., 2008; Neef et al., 2014; Whitesell et al., 2003; Lee et al., 2013; Abravaya et al., 1992; Zou et al., 1998).

Figure 1

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Further regulation of Hsf1 is provided by posttranslational modifications, including phosphorylation, acetylation, sumoylation and oxidation of cysteines to disulfide bridges (Hietakangas et al., 2003; 2006; Sarge et al., 1993; Westerheide et al., 2009; Brunet Simioni et al., 2009; Zhong et al., 1998; Lu et al., 2008). The contribution of these modifications to the primary activating mechanism are still unclear (Budzyński et al., 2015).

To resolve the molecular mechanism of the temperature-induced activation of Hsf1 we analyzed the conformational dynamics of purified monomeric human Hsf1 pretreated at different temperatures using hydrogen-¹H/²H-exchange (HX) mass spectrometry (MS). We found temperature-dependent unfolding of HR-C and concomitant stabilization of HR-A/B, demonstrating that isolated Hsf1 acts as temperature sensor. At short incubation times the temperature response curve exhibits high cooperativity with a transition midpoint of 36°C. Using fluorescence anisotropy we demonstrate that the acquisition of DNA-binding competency depends on temperature and concentration of Hsf1. Phosphomimetic Hsf1 variants corresponding to phosphorylation at two serine residues previously shown to negatively affect Hsf1 activation did not have an increased temperature transition midpoint. Hsp90 known to negatively regulate Hsf1-mediated transcription decreased the slope of the temperature response curve, thereby lowering the transition midpoint and widening the response window. Our data suggest a kinetic model of Hsf1 trimerization.

Recombinant human Hsf1 was purified out of E. coli by affinity chromatography and size-exclusion chromatography, resulting in mostly monomeric species in the final fraction (Figure 1B and C). Upon incubation at 42°C, Hsf1 formed trimers and higher-order oligomers, as verified by blue native gel electrophoresis consistent with published data (Clos et al., 1990), and acquired DNA-binding competence as shown by electrophoretic mobility shift assays (Figure 1C and D). The conformational dynamics of Hsf1 was investigated by HX-MS as described previously (Rist et al., 2006; Graf et al., 2009). Monomeric Hsf1 was incubated for 30 s in D₂O at 20°C, subsequently mixed with ice-cold, low-pH quench buffer to slow down back exchange, and analyzed on our HPLC-mass spectrometry setup including a column with immobilized pepsin for online digestion. As shown in Figure 1E, monomeric Hsf1 is highly dynamic with only few regions exhibiting significant protection from HX, including parts of the DNA binding domain and the trimerization domain (HR-A/B). Out of the C-terminal half of the protein, containing the regulatory region, HR-C and the transactivation domain, only the HR-C region showed significant protection at 20°C consistent with an earlier study showing the C-terminal half of Hsf1 largely unfolded (Pattaramanon et al., 2007). Figure 1F shows a heat map of the DNA binding domain and the trimerization domain of Hsf1, the only parts for which structural information is available.

Hsf1 is a thermosensor

To elucidate temperature-induced changes in conformational dynamics, we pre-incubated monomeric Hsf1 at different temperatures for different time intervals followed by incubation at constant temperature in D₂O (Figure 2A). As control, we analyzed the pre-treated Hsf1 by blue native polyacrylamide gel electrophoresis (Wittig et al., 2006) and observed a temperature-dependent increase in trimeric Hsf1 species (Figure 2B and Figure 2—figure supplement 1). The 10 min-pre-incubation of Hsf1 dramatically changed conformational dynamics of two regions in Hsf1 (Figure 2): temperature-dependent increase in HX is observed in HR-C, indicating heat-induced unfolding, and a concomitant decrease in HX is observed in HR-A/B, consistent with heat-induced trimerization. Close inspection of the spectra of the peptic fragments exhibiting temperature-induced changes in HX revealed bimodal distributions of the isotope clusters indicative of the coexistence of two populations of molecules with different exchange properties (Figure 2—figure supplement 2A and C, Figure 2—figure supplement 3). An equation for two Gaussian curves was fitted to the intensity-versus-m/z plots of the data (Figure 2D and F, and Figure 2—figure supplement 2B and D, Figure 2—figure supplement 3) and the equation parameters used to back calculate the contribution of each population to the peak intensities (see Figure 2—figure supplement 2). For the HR-A/B region the relative frequency of high exchanging population decreases with pre-incubation at increasing temperatures resulting in a sigmoidal temperature response curve (Figure 2E). For the HR-C region the opposite is observed: the frequency of high exchanging species increased with increasing temperatures (Figure 2G). These data clearly demonstrate that Hsf1 has intrinsic thermosensory properties. The midpoint of transition T_m, the temperature at which 50% of the molecules are in the high exchanging conformation after 10 min, was identical for both regions equal to 36.15 ± 0.14°C.

Figure 2 with 4 supplements see all

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We also determined the midpoint of transition for 30 min pre-incubation at different temperatures (Figure 2, Figure 2—figure supplement 3). Under these prolonged incubation conditions, the transition curves became more shallow, and the midpoint temperature for HR-C unfolding and HR-A protection (trimerization) was not identical anymore but decreased to 32.0 ± 0.4 and 34.7 ± 0.2°C, respectively. These data suggest that the temperature-induced conformational changes are not reversible under our conditions, otherwise the steepness of the curves, which is determined by the unfolding enthalpy, should remain the same as for the 10-min-pre-incubation. To investigate this in more detail we heat-shocked Hsf1 for 10 min at 42°C, then incubated the protein for different time intervals at 20°C, and analyzed the conformational state by HX-MS (Figure 3). As control, we incubated Hsf1 without prior heat shock for 30 min at 20°C before HX-MS analysis. We also tested whether dilution would lead to trimer dissociation in the time scale of our experiments (Figure 3G). Both assays clearly demonstrate that HR-C unfolding, HR-A protection and Hsf1 trimerization are not reversible under these conditions. Therefore, we cannot derive the unfolding enthalpy ∆H_U from our temperature response data but only use the fit to determine the temperature at which 50% of the transition occurred.

Figure 3

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Kinetics of temperature-dependent conformational changes

To resolve the kinetics of the conformational transitions, we pre-incubated monomeric Hsf1 for 10-1000 s at 35, 37, 39 or 42°C before diluting into D₂O and incubation for 30 s at 20°C (Figure 4). For the peptic fragments of HR-C (amino acids 378–395 and 389–395) the low-exchanging population decrease with a rate of 0.0028 ± 0.0002, 0.0038 ± 0.0002, 0.011 ± 0.001, and 0.018 ± 0.001 s^-1 at 35, 37, 39 and 42°C, respectively. For the peptic fragments of HR-A/B (amino acids 159–168 and 169–175) the low-exchanging population increased with slightly lower rates of 0.0018 ± 0.0004, 0.0033 ± 0.0002, 0.0094 ± 0.0009 and 0.016 ± 0.001 s^-1 (Figure 4G). The Arrhenius plot of the data yielded the activation energy for the temperature transition of 249 ± 47 kJ·mol^-1 (Figure 4H). Taken together, our data demonstrate that Hsf1 is a thermosensor that directly senses increasing temperatures with conformational changes in HR-A/B and HR-C.

Figure 4

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HR-C interacts with HR-A

To uncouple the temperature-induced HR-C unfolding from trimerization, we replaced the hydrophobic heptad repeat residues in HR-A by serine, which should not engage in coiled-coil interactions. HR-A and HR-B are thought to be involved in trimerization, and HR-B had been previously been implicated in negative regulation of trimerization, since deletion of HR-B lead to continuous active trimeric HSF1 (Zuo et al., 1994). HX-MS experiments with the mutant protein revealed that HR-A/B and HR-C are constitutively unfolded at all temperatures tested (Figure 5). These results demonstrate that the ability of HR-A to form a coiled-coil is essential for stabilization of HR-A, HR-B and HR-C, suggesting that HR-C also interacts with HR-A.

Figure 5

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Mechanism of Hsf1 activation

In our in vitro experiments, the midpoint of trimerization of Hsf1 was around 36°C, which seemed rather low given a body core temperature of 37°C, and DNA binding activity of Hsf1 in different human cells is rather low below 40°C and strongly increased above 42°C (Abravaya et al., 1991; Mosser et al., 1990; Baler et al., 1993). In contrast, in testis the major increase in DNA binding activity of Hsf1 is already observed at 38°C (Sarge, 1995; Sarge et al., 1995). Therefore, the above described unfolding of HR-C cannot be the sole determinant for the setpoint of Hsf1 trimerization and DNA binding activity. Trimerization as multi-molecular reaction would be intrinsically concentration dependent. In contrast, inhibition of trimerization by coiled-coil interaction of HR-C with HR-A/B, as originally proposed by Wu and colleagues (Rabindran et al., 1993), would be an intramolecular reaction and consequently independent of concentration. Thus, the concentration of Hsf1 could be an important parameter for controlling the setpoint of the temperature response curve. Consistent with this hypothesis we noticed spontaneous trimerization of Hsf1 even at 4°C upon concentrating the protein. To explore this hypothesis more quantitatively, we devised a fluorescence anisotropy assay to determine the fraction of Hsf1 capable of binding DNA after treatment at different temperatures. We treated different concentrations of Hsf1 at 30, 35, 37, 39, and 42°C for 10 min, serially diluted the samples, incubated them with fluorescent labeled double-stranded DNA containing heat shock elements and measured fluorescence anisotropy (Figure 6). None of the formed Hsf1 trimers dissociated upon dilution and incubation at room temperature (Figure 3G). If Hsf1 trimerization were not concentration dependent in the concentration range tested, all data points would fall onto the same titration curve. This was obviously not the case. The quadratic solution of the binding equilibrium, modified for fractional active protein was globally fitted to the data, assuming identical K_D values for all formed Hsf1 trimers and similar minimum and maximum anisotropy values for no binding and complete binding, respectively. This fit yielded the fraction of DNA binding competent Hsf1 trimers. For 100 nM Hsf1, no activation was observed up to 42°C (Figure 6F). At 300 nM, less than 20% of the theoretical possible Hsf1 trimers had formed at 42°C within 10 min. In contrast, at 1 and 5 µM concentrations a substantial fraction of Hsf1 trimers had formed already at lower temperatures. At 42°C the apparent fraction of DNA-binding competent Hsf1 species may decrease due to the formation of high order oligomers as observed by blue native gel (Figure 1C).

Figure 6

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Since these data indicated that the temperature transition of Hsf1 is concentration dependent, we wondered whether only trimerization is affected or HR-C unfolding as well. We therefore repeated the HX-MS experiments at lower concentration (2 µM). Interestingly, not only trimerization but also unfolding of HR-C was concentration dependent and the differences between the transition temperatures of HR-A/B and HR-C were not statistically significant (Figure 7B). However, the difference in transition temperature between Hsf1 at 5 µM and at 2 µM was highly statistically significant (p<0.0001). We could not test lower concentrations of Hsf1 due to lacking sensitivity in the mass spectrometric detection of the important peptides.

Figure 7

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Taken together, these data clearly demonstrate that HR-C unfolding, trimerization and the fraction of DNA-competent Hsf1 trimers are a function of temperature and concentration.

Influence of Hsp90 on Hsf1 activation

Several lines of evidence suggested that Hsp90 inhibits Hsf1 activation (Zou et al., 1998) and the current model assumes that Hsp90 binds Hsf1 in the monomeric state in unstressed cells (Anckar and Sistonen, 2011). We therefore studied the effect of human Hsp90β on the conformational dynamics of Hsf1, in particular the temperature response curve (Figure 8A–C). Surprisingly, in the presence of Hsp90 the midpoint of transition was lower than in its absence and the response curve was less steep, stretching the transition window from ~10° in the absence of Hsp90 to ~20°. Interestingly, the midpoint temperature of transition in the presence of Hsp90 was slightly lower for the peptide derived from HR-C than for the HR-A/B peptides (Figure 8G). A significant difference in midpoint temperature for the two regions was never observed in other experiments with 10-min-pre-incubation time but with a 30-min-pre-incubation at the different temperatures (compare Figure 8G with Figure 7B).

Figure 8

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To verify that the effect of Hsp90 on trimerization is not an artifact of HX-MS methodology we performed electrophoretic mobility shift assays under comparable conditions using a fluorescently labeled DNA probe containing heat shock elements (Figure 8D–F). Within the experimental error, the results of the DNA binding assay were identical to those of the HX-MS experiments (Figure 8G). Taken together, our results demonstrate that in an in vitro assay with purified components, Hsp90 neither inhibits Hsf1 trimerization nor its DNA binding but, on the contrary, lowers transition temperature and widens the activation window.

In this study we demonstrate that human Hsf1 is a thermosensor. We are the first to show that the HR-C region of Hsf1 unfolds with temperature-dependent rates, resulting in a release of its repressive effect on Hsf1 trimerization and DNA binding. For relatively short heat shocks unfolding of HR-C and trimerization through intermolecular interactions of HR-A/B exhibit the same temperature response curves and follow identical kinetics, suggesting that these are coupled events. For longer heat shocks the cooperativity of the transition is reduced and HR-C unfolding and HR-A/B trimerization seem to uncouple. Most importantly, HR-C unfolding, temperature-induced trimerization and acquisition of DNA-binding competence depends on the concentration of Hsf1. Moreover, Hsp90 significantly modulated the temperature response of Hsf1, reducing midpoint and steepness of the response curve, thus widening the temperature window within which Hsf1 transits from monomer to trimer and low to maximal DNA binding competence. The response curves of Hsf1 in the presence of Hsp90 are similar to the response curves at prolonged incubation time at elevated temperatures. Thus, Hsp90 accelerates the response at intermediate heat shock conditions.

Our data with purified human Hsf1 are consistent with previous in vivo and in vitro work (Rabindran et al., 1993; Zhong et al., 1998; Baler et al., 1993; Zuo et al., 1994; 1995; Sarge et al., 1993; Zou et al., 1998), substantiating the hypothesis that Hsf1 trimerization and DNA binding is controlled by HR-C. Most previous work was performed in cellular systems or complex cell extracts and only few studies used purified components. Purified Drosophila Hsf1 was shown to exist in a trimer-monomer equilibrium that was influenced by temperature and oxidative stress (Zhong et al., 1998). However, human HSF1 does not seem to exist in such an equilibrium, as we did not observe dissociation of trimeric human Hsf1 upon dilution and heat-induced trimerization was irreversible in our hands (Figure 3). These data suggest that there are principle differences between Drosophila and human Hsf1.

For human Hsf1 we observed striking differences between the temperature response curves of 10 and 30-min-incubation at elevated temperatures. How can these differences be explained? Under our conditions temperature-induced Hsf1 transitions were irreversible. Therefore, at low temperatures even rare unfolding fluctuations of HR-C will eventually lead to trimerization, which will not be observed at short incubation times. Two effects could be responsible for the difference in T_m for HR-A/B and HR-C. At low temperature HR-C dissociation from HR-A/B and re-association might be fast as compared to trimerization. In the free state, HR-C could exist in an unfolding-refolding equilibrium, allowing exchange of protons for deuterons. If refolding is slow in comparison to the intrinsic chemical exchange rate, this would be visible as unfolded species, but HR-C would still refold and reassociate with HR-A/B, repressing trimerization. With increasing temperature unfolding rates would increase, shifting the equilibrium to the completely unfolded state, then allowing HR-A/B trimerization. Alternatively, additional temperature-induced conformational changes in HR-A/B are necessary to allow trimerization and these changes are slow as compared to HR-C unfolding at low temperatures.

Phosphorylation of Ser307 was suggested to repress human Hsf1 activation because the Ser307 to Ala replacement caused constitutively active Hsf1 in vivo (Xia et al., 1998). The temperature response curve of the phosphomimetic Hsf1-S307D variant measured by HX-MS showed a slightly reduced midpoint of transition as compared to wild-type Hsf1, suggesting that phosphorylation at this site does not inhibit heat-induced trimerization but might rather favor it and the repressive effect must be at a different level. Our observations are consistent with more recent data on non-phosphorylatable Hsf1 variants which were not constitutively active, casting a doubt on the repressive effect of phosphorylation at this site (Budzyński et al., 2015).

Most surprising was our finding that Hsp90 does not prevent trimerization and DNA binding of Hsf1 but in the contrary reduces midpoint and steepness of the temperature response curve. This observation seems to be at odds with the known repressive function of Hsp90 on the heat shock response (Zou et al., 1998; Whitesell et al., 2003; 2014; Sittler et al., 2001; Ali et al., 1998). This discrepancy may have different reasons. First, Hsp90 also inhibits the heat shock response in yeast, although yeast Hsf1 is constitutively trimeric and bound to DNA, suggesting that Hsp90 could exert its inhibiting function on human Hsf1 after trimerization and DNA binding as well, consistent with observations for human Hsf1 (Duina et al., 1998; Sorger et al., 1987; Guo et al., 2001). Second, in our experiments we only used one isoform of Hsp90, Hsp90β, and did not add any of the some 30 co-chaperones known to assist chaperoning by Hsp90. Further experiments with Hsp90α and different combinations of co-chaperones will be necessary to elucidate whether there exist isoform specificity in Hsf1 regulation or whether Hsp90-co-chaperone complexes have a different effect on Hsf1 trimerization than Hsp90β alone. Third, effects of Hsp90 down-regulation or inhibition could also be indirect, especially because interaction of Hsp90 with Hsf1 seems only to be observed after cross-linking (Neef et al., 2014; Zou et al., 1998), suggesting a very transient interaction. Hsp90 chaperones many kinases, and inhibition of Hsp90 leads to inactivation and degradation of these client proteins. Loss of such kinases could reduce inhibitory effects of phosphorylation or phosphorylation-dependent sumoylation of Hsf1 (Soncin et al., 2003; Wang et al., 2006; Hietakangas et al., 2003; 2006).

Originally, it was proposed that HR-C forms a coiled-coil with HR-A or HR-B to prevent trimerization in unstressed HSF1 (Rabindran et al., 1993; Zuo et al., 1994). Together with the observation that Hsf1 spontaneously trimerizes at high concentrations in the absence of a heat shock ([Zhong et al., 1998] and our own observations) and with our HX-MS data, showing temperature-induced unfolding of HR-C, the model shown in Figure 9A can be derived. Under non-stress conditions, HSF1 is in a conformational equilibrium between a closed conformation with HR-C interacting with HR-A/B and an open conformation, in which the two heptad repeat regions are dissociated. Since association of HR-C with HR-A/B is an intramolecular interaction, limiting the diffusional freedom of the interaction partners, association rates would be very high due to the apparent high local concentration. In addition, the net charge of HR-A/B and HR-C are +6 and -7, respectively, favoring association of the two regions by electrostatic attraction. Only at high concentrations of HSF1 association of HR-A/B regions of several HSF1 molecules, which are in the open conformation, would be able to compete with the intramolecular reaction, forming the thermodynamically more stable trimer. This would explain the spontaneous Hsf1 trimerization at high concentrations even at low temperatures (Zhong et al., 1998). Temperature-induced unfolding of HR-C in the closed conformation favors HR-C dissociation. Alternatively or in addition, HR-C unfolding in the open conformation prevents coiled-coil interaction with HR-A/B and thus reduces the back-reaction to the closed conformation. As a consequence, trimer association is favored even at lower HSF1 concentrations.

Figure 9

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For two reasons we do not consider this model as very likely: First, single helices free in solutions are usually not stable but are in a rapid equilibrium with the unfolded state due to the low energy difference between helix-internal hydrogen bonds and hydrogen bonds with water. In such a state, we would not expect to see much protection in HX-MS experiments, which is in contrast to our observation (Figure 1E). The amount of heat necessary to unfold a single helix seems too small to account for the temperature control, the steepness, the kinetics and the substantial activation energy of 249 kJ·mol^-1 for the unfolding/trimerization transition, observed in our experiments. Second, HR-C unfolding would be independent of concentration in this model, also inconsistent with our data (Figure 7).

Based on our data we propose a novel ‘dimer activation model’ (Figure 9B). In this model HR-C remains bound to HR-A/B in the unstressed Hsf1 monomer. However, unstressed Hsf1 could transiently dimerize due to the larger size of the HR-A/B region (75 amino acids) as compared to HR-C (42 amino acids). Such dimers would be destabilized by the interaction of HR-C with HR-A/B, resulting in high dissociation rates and a high fraction of Hsf1 monomers in unstressed cells. However, HR-A/B-HR-A/B interaction could also destabilize the interaction of HR-C with HR-A/B. At high Hsf1 concentrations, association of a third Hsf1 monomer to the transient Hsf1 dimer could occur, displacing the then more-weakly bound HR-C and leading to a stable Hsf1 trimer. Heat-induced unfolding of HR-C and possibly also its binding partner within HR-A/B (in our experiments obscured by subsequent trimerization) would lead to HR-C undocking. This would reduce Hsf1 dimer dissociation rates and favor Hsf1 trimerization even at low Hsf1 concentrations.

In this model, HR-C unfolding and trimerization are kinetically coupled processes, at least for short heat shocks (up to 15 min), explaining why they occur at identical rate constants (Figure 4). Furthermore, the average energy to unfold a protein which does not contain a co-factor is 1.4 kJ·mol^-1 (Privalov and Gill, 1988; Alexander et al., 1992). Dividing the activation energy for HR-C unfolding determined by us, 249 kJ·mol^-1, by 1.4 results in 175, suggesting that 175 residues are involved in this unfolding process. This is close to 168, the number of residues corresponding to a dimer of the coiled-coil between HR-C (42 residues) with a similar sized region in HR-A/B.

Our dimer activation model assumes that the proposed Hsf1 dimer has a lower T_m (T_m,D) than the Hsf1 monomer and that the measured T_m depends on the fraction of Hsf1 dimer present in the assay. We therefore plotted all of our T_m values derived for wild-type Hsf1 and incubation times of 10 min versus Hsf1 concentration and fitted the quadratic solution of the law of mass action for the monomer-dimer equilibrium to the data (Figure 9C). This fit results in a K_D for the monomer-dimer equilibrium, the T_m,M for the monomer and the T_m,D for the dimer. However, the derived values are only very rough estimates due to the sensitivity of the curve to values of very low concentrations of Hsf1, which, for technical reasons, we could not determine so far. However, the steepness of the curve demonstrates that already small changes in concentration can dramatically change the transition temperature for Hsf1 activation.

Although there are no concentration determinations for Hsf1 in different tissues available to our knowledge, using the relative quantification data determined by mass spectrometry for 11 different cancer cell lines (Geiger et al., 2012), and assuming that these cells have a total protein concentration of about 150 mg/ml as determined for HEK293 cells (Gillen and Forbush, 1999), results in Hsf1 concentrations between 10 and 130 nM. The resulting T_m values would be between 47 and 53°C. However, Hsf1 is not equally distributed throughout the cytosol but shuttles in and out of the nucleus with various stress conditions preventing Hsf1 export out of the nucleus (Vujanac et al., 2005), resulting in a locally increased Hsf1 concentration between 4- and 17-fold (Fujioka et al., 2006) and a local concentration between 40 and 2210 nM. This is well within the range that would lead to a functional heat shock response according to our model.

Interestingly, the recent crystal structure of the trimerization domain of the Hsf1 homolog Skn7 of Chaetomium thermophilum contains trimers but also tetramers (PDB ID 5D5Z and 5D5Y, [Neudegger et al., 2016]). This tetramer might be a proxy for the dimer of HR-C-HR-A/B coiled-coils. To visualize how such an Hsf1 dimer might look like, we modeled the structure of HR-A, HR-B and HR-C using I-TASSER (Roy et al., 2010; Zhang, 2008; Yang and Zhang, 2015; Yang et al., 2015) and the tetramer structure of the trimerization domain of Skn7 as template (Figure 9D).

Hsp90 could modulate the monomer-trimer transition by stabilizing HR-A-HR-A interactions and/or destabilize HR-A/B-HR-C interactions, resulting in a reduced dimer dissociation rate and an increased rate of trimerization at lower temperatures. Stabilization of the HR-A-HR-A dimer and concomitant destabilization of HR-A/B-HR-C interaction would automatically destabilize HR-C ,since single helices are not stabile in solution and only stabilized by interaction with other structural elements, leading to a reduced unfolding transition temperature. To distinguish between stabilization of HR-A-HR-A interaction and destabilization of HR-A/B-HR-C interactions we simulated the effect of Hsp90 on the T_m by varying the apparent K_D for dimerization and/or the T_m,D of the dimer (see Figure 9C, dotted lines). With the current data available only changing the K_D does not reduce the T_m sufficiently to fit the measured values. However, small changes in K_D and T_m,D would give satisfying results. This would also explain the observation that Hsp90 reduced the T_m for HR-C unfolding significantly more than the T_m for trimerization. In this respect Hsp90, curiously, had a similar effect as the prolonged incubation at elevated temperatures (compare transition curves in Figure 2E and G and Figure 8B, C, and F). Thus, Hsp90 accelerates temperature-induced changes in conformation of Hsf1. It is not surprising that the chaperone Hsp90 destabilizes the HR-C conformation. Chaperones have been shown to locally unfold native proteins (Rodriguez et al., 2008; Sharma et al., 2010; Kirschke et al., 2014) and Hsp90 is believed to destabilize an α-helix in steroid hormone receptors to allow hormone binding.

How could Hsf1 be active at non-heat stress conditions, for example during development (Xiao et al., 1999), and how could it be activated by salicylate, low pH, Ca²⁺ ions, hypoxia, or proteotoxic stress other than heat shock as demonstrated previously (Mosser et al., 1990; Jurivich et al., 1992; Huang et al., 1995; Liu et al., 1996; Zhong et al., 1999; Ahn and Thiele, 2003)? According to our model Hsf1 exists in a monomer-dimer equilibrium, and trimerization of Hsf1 with subsequent DNA binding may occur continuously at low levels, promoted by Hsp90, as shown by us, and inhibited by TriC/CCT (Neef et al., 2014). This may ensure basal Hsf1 transcriptional activity under non-stress conditions. At the same time inhibition of Hsf1 activity by Hsp90, Hsp70-mediated attenuation and continuous Hsf1 monomerization would keep heat shock gene transcription at a low level. Any condition that would favor Hsf1 dimerization and thus trimerization or inhibit chaperone-mediated inhibition or attenuation would, as a consequence, increase heat shock gene transcription. All of the conditions mentioned above, including development, have been associated with an imbalance in proteostasis affecting Hsf1 through titrating away chaperones. However, Hsf1 dimerization could also be affected directly by changing its local concentration, as through transport of Hsf1 into the nucleus (Dai et al., 2003) or preventing its export (Vujanac et al., 2005), and by posttranslational modifications, including glutathionylation of the cysteine in HR-A in response to oxidative stress or alkylating agents (Liu et al., 1996), and phosphorylation of Thr142 in HR-A (Soncin et al., 2003), both of which would reduce the positive net charge of HR-A and thus the electrostatic repulsion.

Our model could explain why the temperature setpoint of activation was lower when human Hsf1 was expressed in Drosophila cells or in Xenopus oocytes (Baler et al., 1993; Clos et al., 1993). In transient or stable transfection experiments, usually a strong promoter is used to express the transfected gene. Thus, the concentration of Hsf1 might have been much higher in the transfected cell than is naturally the case in human cells. Similarly, in Xenopus oocyte-injection experiments the amount of injected mRNA determines the final concentration of HSF1 and might have been so high that the resulting Hsf1 concentration might have allowed activation already at 37°C. Finally, our kinetic Hsf1 activation model would allow each cell to adjust its setpoint of activation by changing the concentration of Hsf1 by producing more Hsf1 or by concentrating it in a smaller compartment, for example by transport from the cytoplasm into the nucleus. This would easily explain the different setpoints in testis (Sarge et al., 1995; Sarge, 1995), mouse T-lymphocytes (Gothard et al., 2003) and mouse motor neurons (Batulan et al., 2003). This might be particularly important for cancer cells for which it was shown that Hsf1 is a driver of malignancy (Dai et al., 2007).

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Author details

1. Nikolai Hentze

   Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany

   Present address

   NBE Analytical R&D, AbbVie Deutschland GmbH & Co. KG, Ludwigshafen, Germany

   Contribution

   NH, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Laura Le Breton

   Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany

   Contribution

   LLB, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Jan Wiesner

   Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany

   Contribution

   JW, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Georg Kempf

   Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany

   Present address

   Biochemiezentrum, Universität Heidelberg, Heidelberg, Germany

   Contribution

   GK, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. Matthias P Mayer

   Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany

   Contribution

   MPM, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   M.Mayer@zmbh.uni-heidelberg.de

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-7859-3112

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