The role of environment has been contemplated in theories of evolution for over a hundred years (Darwin, 1859; Darwin and Wallace, 1858; Wright, 1932), yet molecular level analyses of how environment impacts the evolution of gene sequences remain experimentally under-explored. Depending on environmental conditions, mutations can be categorized into three classes: strongly deleterious mutations that are purged from populations by purifying selection, nearly-neutral mutations that are governed by stochastic processes, and beneficial mutations that tend to provide a selective advantage (Ohta, 1973). It has long been clear that environmental conditions can alter the fitness effects of mutations (Tutt, 1896). However, examining how environmental conditions impact any of the three classes of mutations is challenging. Measurable properties of nearly-neutral and deleterious mutations in natural populations are impacted by both demography and selection (Ohta, 1973), which are difficult to disentangle. In addition, many traits are complex, making it challenging to identify all contributing genetic variations (McCarthy et al., 2008). For these and other reasons, we do not have a detailed understanding of how environmental conditions impact the evolution of most gene sequences.

Mutational scanning approaches (Fowler et al., 2010) provide novel opportunities to examine fitness effects of the same mutations under different laboratory conditions (Boucher et al., 2016; Boucher et al., 2014; Canale et al., 2018; Kemble et al., 2019). The EMPIRIC (Exceedingly Meticulous and Parallel Investigation of Randomized Individual Codons) approach that we previously developed (Hietpas et al., 2011) is particularly well suited to address questions regarding the environmental impact of mutational effects for three reasons: it quantifies growth rates that are a direct measure of experimental fitness, all point mutations are engineered providing comprehensive maps of growth effects, and all the variants can be tracked in the same flask while experiencing identical growth conditions. We have previously used the EMPIRIC approach to investigate how protein fitness maps of ubiquitin vary in different environmental conditions (Mavor et al., 2016). The analysis of ubiquitin fitness maps revealed that stress environments can exacerbate the fitness defects of mutations. However, the small size of ubiquitin and the near absence of natural variation in ubiquitin sequences (only three amino acid differences between yeast and human) hindered investigation of the properties underlying historically observed substitutions.

Mutational scanning approaches have emerged as a robust method to analyze relationships between gene sequence and function, including aspects of environmental-dependent selection pressure. Multiple studies have investigated resistance mutations that enhance growth in drug or antibody environments (Dingens et al., 2019; Doud et al., 2018; Firnberg et al., 2014; Jiang et al., 2016; Stiffler et al., 2015). Most of these studies have focused on interpreting adaptation in the light of protein structure. Of note, Dandage, Chakraborty and colleagues explored how environmental perturbations to protein folding influenced tolerance of mutations in the 178 amino acid gentamicin-resistant gene in bacteria (Dandage et al., 2018). However, the question of how environmental variation shapes the selection pressure on gene sequences has not been well studied.

Here, we report comprehensive experimental fitness maps of Heat Shock Protein 90 (Hsp90) under multiple stress conditions and compare our experimental results with the historical record of hundreds of Hsp90 substitutions accrued during its billion years of evolution in eukaryotes. Hsp90 encodes a 709 amino acid protein and to our knowledge it is the largest gene for which a comprehensive protein fitness map has been determined. Hsp90 is an essential and highly abundant molecular chaperone which is induced by a wide variety of environmental stresses (Gasch et al., 2000; Lindquist, 1981). Hsp90 assists cells in responding to these stressful conditions by facilitating the folding and activation of client proteins through a series of ATP-dependent conformational changes mediated by co-chaperones (Krukenberg et al., 2011). These clients are primarily signal transduction proteins, highly enriched in kinases and transcription factors (Taipale et al., 2012). Through its clients, Hsp90 activity is linked to virtually every cellular process.

Hsp90 can facilitate the emergence and evolution of new traits in response to stress conditions, including drug resistance in fungi (Cowen and Lindquist, 2005), gross morphology in flies (Rutherford and Lindquist, 1998) and plants (Queitsch et al., 2002), and vision loss in cave fish (Rohner et al., 2013). In non-stress conditions, an abundance of Hsp90 promotes standing variation by masking the phenotypic effects of destabilizing mutations in clients. Stressful conditions that tax Hsp90 capacity can then manifest in phenotypic diversity that can contribute to adaptation. Because of the biochemical and evolutionary links between Hsp90 and stress, we hypothesized that environmental stress would result in altered fitness maps.

The conditions in natural environments often fluctuate, and all organisms contain stress response systems that aid in acclimation to new conditions. The conditions experienced by different populations can vary tremendously depending on the niches that they inhabit, providing the potential for distinct selective pressures on Hsp90. Previous studies of a nine amino acid loop in Hsp90 identified multiple amino acid changes that increased the growth rate of yeast in elevated salinity (Hietpas et al., 2013), demonstrating the potential for environmental-dependent beneficial mutations in Hsp90. However, the sequence of Hsp90 is strongly conserved in eukaryotes (57% amino acid identity from yeast to human), indicating consistent strong purifying selection.

To investigate the potential influence of the environment on Hsp90 evolution, we quantified fitness maps in six different conditions. While proximity to ATP is the dominant functional constraint in standard conditions, the influence of client and co-chaperone interactions on growth rate dramatically increases under stress conditions. Increased selection pressure from heat and diamide stresses led to a greater number of beneficial variants compared to standard conditions. The observed beneficial variants were enriched at functional hotspots in Hsp90. However, the natural variants of Hsp90 tend to support efficient growth in all environments tested, indicating selection for robustness to diverse stress conditions in the natural evolution of Hsp90.

We developed a powerful experimental system to analyze the growth rate supported by all possible Hsp90 point mutations under distinct growth conditions. Bulk competitions of yeast with a deep sequencing readout enabled the simultaneous quantification of 98% of possible amino acid changes (Figure 1A). The single point mutant library was engineered by incorporating a single degenerate codon (NNN) into an otherwise wild-type Hsp90 sequence as previously described (Hietpas et al., 2012). To provide a sensitive readout of changes in Hsp90 function, we transferred the library to a plasmid under a constitutive low-expression level ADH promoter that reduced Hsp90 protein levels to near-critical levels (Jiang et al., 2013). To efficiently track all variants in a single competition flask so that all variants experience identical conditions, we updated our previously developed EMPIRIC approach to include a barcoding strategy (Hietpas et al., 2012). As described in the Materials and methods, this barcode strategy enabled us to track mutations across a large gene using a short sequencing readout.

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Sequencing counts and selection coefficients for each individual amino acid change across amino acids 2–709 of Hsp90 in both replicates of standard conditions.

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Average selection coefficient (excluding stops) at each position of Hsp90 in Standard replicate 1.

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We transformed the plasmid library of comprehensive Hsp90 point mutations into a conditional yeast strain where we could turn selection of the library on or off. We used a yeast Hsp90-shutoff strain in which both paralogs of Hsp90 (hsc82 and hsp82) are deleted and a copy of hsp82 with expression under strict regulation of a galactose-inducible promoter is integrated into the chromosome (Jiang et al., 2013). The mutant libraries were amplified in the absence of selection on the mutant variant by growing the transformed yeast in galactose media that expresses the wild-type chromosomal copy of hsp82. We switched the yeast to dextrose media to shut off the expression of wild-type Hsp90, allowing the mutagenized variants to be the sole source of Hsp90 protein in the cell, and then split the culture into six different environmental conditions. We extracted samples from each condition at multiple time points and used Illumina sequencing to estimate the frequency of each Hsp90 variant over time. We assessed the selection coefficient of each Hsp90 variant from the change in frequency relative to wild-type Hsp90 using a previously developed Bayesian Markov Chain Monte Carlo (MCMC) method (Bank et al., 2014; Fragata et al., 2018), where 0 represents wild-type and −1 represents null alleles (Figure 1—source data 1).

To analyze reproducibility of the growth competition, we performed a technical replicate under standard conditions. We used a batch of the same transformed cells that we had frozen and stored such that the repeat bulk competition experiments and sequencing were performed independently. Selection coefficients between replicates were strongly correlated (R² = 0.90), and indicated that we could clearly distinguish between selection coefficients for members of the library containing silent mutations that do not change the amino acid sequence (wild-type synonyms) and those containing stop codons (Figure 1B, Figure 1—figure supplement 1). For the second replicate we noted a small fitness defect (s ~ –0.2) for wild-type synonyms at positions 679–709 relative to other positions (Figure 1—figure supplement 1). We did not see this behavior in any other condition or replicate tested and do not understand its source. The selection coefficients in this study under standard conditions also correlated strongly (R² = 0.87) with estimates of the Hsp90 N-domain in a previous study (Mishra et al., 2016; Figure 1—figure supplement 2), indicating that biological replicates also show high reproducibility. Of note, variants with strongly deleterious effects exhibited the greatest variation between replicates, consistent with the noise inherent in estimating the frequency of rapidly depleting variants (Figure 1B). The stop codons were already partially depleted from the cells at the 0 time point, likely contributing to their variation between replicates (Figure 1—figure supplement 3A). In accordance with this, there was a higher variation in selection coefficients between replicates for stop codons with the lowest initial reads (Figure 1—figure supplement 3B). Stop codon fitness was similar for all three stop codons (Figure 1—figure supplement 3C) and at positions across Hsp90 with exception of the last 32 positions that have previously been shown to be dispensable for its viability (Louvion et al., 1996; Figure 1—figure supplement 1). A heatmap representation of all the selection coefficients determined in standard conditions in replicate one is shown in Figure 1—figure supplement 4.

The large number of signaling pathways that depend on Hsp90 (Taipale et al., 2012) and its strong sequence conservation suggest that many mutations of Hsp90 may decrease fitness. However, most variants of Hsp90 had wild-type-like fitness in the competition experiment in standard conditions (Figure 1C, Figure 1—figure supplement 4). All possible mutations (excluding stops) were compatible with function at 425 positions. Only 17 positions had low mutational tolerance to the extent that 15 or more substitutions caused null-like growth defects (R32, E33, N37, D40, D79, G81, G94, I96, A97, S99, G118, G121, G123, Y125, F156, W300, and R380). All these positions except for W300 are in contact with ATP or mediate ATP-dependent conformational changes in the N-domain of Hsp90. In fact, the average selection coefficient at different positions (a measure of mutational sensitivity) in standard growth conditions correlates (R² = 0.49) with distance from ATP (Figure 1—figure supplement 5). While W300 does not contact ATP, it transmits information from client binding to long range conformational changes of Hsp90 that are driven by ATP hydrolysis (Röhl et al., 2013). Our results indicate that ATP binding and the conformational changes driven by ATP hydrolysis impose dominant physical constraints in Hsp90 under standard laboratory conditions.

At first sight, the observation that most mutations are compatible with robust growth in standard conditions is at odds with the fact that the Hsp90 sequence is strongly conserved across large evolutionary distances (Figure 1C). One potential reason for this discrepancy could be that the strength of purifying selection in large natural populations over long evolutionary time-scales is more stringent than can be measured in the laboratory. In other words, experimentally unmeasurable fitness defects could be subject to purifying selection in nature. In addition, the range of environmental conditions that yeast experience in natural settings may not be reflected by standard laboratory growth conditions. To investigate the impact of environmental conditions on mutational effects in Hsp90, we measured the growth rate of Hsp90 variants under five additional stress conditions.

Impact of stress conditions on mutational sensitivity of Hsp90

We measured the fitness of Hsp90 variants in conditions of nitrogen depletion (ND) (0.0125% ammonium sulfate), hyper-osmotic shock (0.8 M NaCl), ethanol stress (7.5% ethanol), the sulfhydryl-oxidizing agent diamide (0.85 mM), and temperature shock (37°C). All these stresses are known to elicit a common shared environmental stress response characterized by altered expression of ~900 genes as well as having specific responses unique to each stress (Gasch et al., 2000). Genes encoding heat-shock proteins, including Hsp90, are transiently upregulated in all these stresses except elevated salinity (Gasch et al., 2000; Piper, 1995).

One way to characterize stress conditions is to measure the extent to which they slow down growth. For our experiments, each of the environmental stresses were selected to partially decrease the growth rate. Consistently, all stresses reduced the growth rate of the parental strain within a two-fold range, with depletion of nitrogen levels causing the smallest reduction in growth rate and diamide causing the greatest reduction (Figure 2A). To investigate how critical Hsp90 is for growth in each condition, we measured growth rates of yeast with either normal or more than 10-fold reduced (Jiang et al., 2013) levels of Hsp90 protein (Figure 2A). Under standard conditions, the normal level of Hsp90 protein can be dramatically reduced without major impacts on growth rate, consistent with previous findings (Jiang et al., 2013; Picard et al., 1990).

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Sequencing counts and selection coefficients for each individual amino acid change across amino acids 2–709 of Hsp90 in Nitrogen Depletion, Salt, Ethanol, Diamide and 37°C.

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We anticipated that Hsp90 would be required at increased levels for robust experimental growth in diamide, nitrogen starvation, ethanol, and high temperature (Gasch et al., 2000) based on the concept that cells increase expression level of genes in conditions where those gene products are needed at higher concentration. Consistent with this concept, reduced Hsp90 levels cause a marked decrease in growth rate at 37°C. However, Hsp90 protein levels had smaller impacts on growth rates under the other stress conditions, indicating that reliance on overall Hsp90 function does not increase dramatically in these conditions.

We quantified the growth rates of all Hsp90 single-mutant variants in each of the stress conditions as selection coefficients (Figure 2—source data 1, Figure 2—figure supplements 1–5). We could clearly differentiate between the selection coefficients of wild-type synonyms and stop codons in all conditions (Figure 2B, Figure 2—figure supplement 6), and we normalized to these classes of mutations to facilitate comparisons between each condition (Figure 2—figure supplement 7). Of note, the observed selection coefficients of wild-type synonyms varied more in conditions of high temperature and diamide stress compared to standard (Figure 2—figure supplement 8A,B). We also note greater variation in the selection coefficients of barcodes for the same codon in the diamide and high temperature conditions (Figure 2—figure supplement 8C). We conclude that diamide and elevated temperature provided greater noise in our selection coefficient measurements. To take into account differences in signal to noise for each condition, we either averaged over large numbers of mutations or categorized selection coefficients as wild-type-like, strongly deleterious, intermediate, or beneficial based on the distribution of wild-type synonyms and stop codons in each condition (see Materials and methods and Figure 2—figure supplement 7).

We compared selection coefficients of each Hsp90 variant in each stress condition to standard condition (Figure 2B&C). The stresses of 37°C and diamide tend to exaggerate the growth defects of many mutants compared to standard conditions, whereas high salt and ethanol tend to rescue growth defects (Figure 2B&C and Figure 2—figure supplement 9). According to the theory of metabolic flux (Dykhuizen et al., 1987; Kacser and Burns, 1981), gene products that are rate limiting for growth will be subject to the strongest selection. Accordingly, the relationship between Hsp90 function and growth rate should largely determine the strength of selection acting on Hsp90 sequence. Conditions where Hsp90 function is more directly linked to growth rate would be more sensitive to Hsp90 mutations than conditions where Hsp90 function can be reduced without changing growth rates (Bershtein et al., 2013; Jiang et al., 2013). The average selection coefficients are more deleterious in diamide and temperature stress compared to standard conditions. These findings are consistent with heat and diamide stresses causing a growth limiting increase in unfolded Hsp90 clients that is rate limiting for growth. In contrast, the average selection coefficients are less deleterious in ethanol and salt stress than in standard conditions, which suggests a decrease in the demand for Hsp90 function in these conditions. Due to the complex role Hsp90 plays in diverse signaling pathways in the cell, the different environmental stresses may differentially impact subsets of client proteins that cause distinct selection pressures on Hsp90 function.

Structural analyses of environmental responsive positions

Altering environmental conditions had a pervasive influence on mutational effects along the sequence of Hsp90 (Figure 3A and Figure 3—figure supplement 1). We structurally mapped the average selection coefficient of each position in each condition relative to standard conditions as a measure of the sensitivity to mutation of each position under each environmental stress (Figure 3A, Figure 3—source data 1). Many positions had mutational profiles that were responsive to a range of environments. Environmentally responsive positions with large changes in average selection coefficient in at least three conditions are highlighted on the Hsp90 structure in green in Figure 3B. Unlike the critical positions that cluster around the ATP binding site (Figure 1C), the environmentally responsive positions are located throughout all domains of Hsp90. Similar to critical residues, environmentally responsive positions are more conserved in nature compared to other positions in Hsp90 (Figure 3C), suggesting that the suite of experimental stress conditions tested captured aspects of natural selection pressures on Hsp90 sequence.

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Average selection coefficient (excluding stops) at each position of Hsp90 in each environmental condition relative to the average selection coefficient in standard conditions.

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Hsp90 positions with environmentally responsive selection coefficients were enriched in binding contacts with clients, co-chaperones and intramolecular Hsp90 contacts involved in transient conformational changes (Figure 3D and Figure 3—figure supplement 2A). About 65% of the environmentally responsive residues have been identified either structurally or genetically as interacting with binding partners (Ali et al., 2006; Bohen and Yamamoto, 1993; Genest et al., 2013; Hagn et al., 2011; Hawle et al., 2006; Kravats et al., 2018; Lorenz et al., 2014; Meyer et al., 2003; Meyer et al., 2004; Nathan and Lindquist, 1995; Retzlaff et al., 2009; Roe et al., 2004; Verba et al., 2016; Zhang et al., 2010), compared to about 15% of positions that were not responsive to stress conditions. This analysis was performed on the small subset of clients and cochaperones with known Hsp90-binding sites. While ATP binding and hydrolysis are the main structural determinants that constrain fitness in standard growth conditions, client and co-chaperone interactions have a larger impact on experimental fitness under stress conditions. Although the mean selection coefficients of mutations at the known client and co-chaperone binding sites are responsive to changes in environment, the direction of the shift of growth rate compared to standard conditions depends on the specific binding partner and environment (Figure 3E and Figure 3—figure supplement 2B). This suggests that different environments place unique functional demands on Hsp90 that may be mediated by the relative affinities of different clients and co-chaperones. Consistent with these observations, we hypothesize that Hsp90 client priority is determined by relative binding affinity and that Hsp90 mutations can reprioritize clients that in turn impacts many signaling pathways.

Constraint of mutational sensitivity at high temperature

We find that different environmental conditions lead to distinct selection on Hsp90 based on the number of beneficial and deleterious variants in each condition, including elevated temperature placing the greatest purifying selection pressure on Hsp90. Of the 2504 variants of Hsp90 that are deleterious when grown at 37°C, 884 of them (~35%) are deleterious only in this condition (Figure 3F). We defined mutants that confer temperature sensitive (ts) growth phenotypes on cells as variants with selection coefficients within the distribution of wild-type synonyms in standard conditions and that of stop codons at 37°C. Based on this definition, 663 Hsp90 amino acid changes (roughly 5% of possible changes) were found to be temperature sensitive (Figure 4A, Figure 4—source data 1). We sought to understand the physical underpinnings of this large set of Hsp90 ts mutations.

Figure 4

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List of all temperature-sensitive mutants and associated selection coefficients.

Ts mutants were defined as variants with selection coefficients within the distribution of wild-type synonyms in standard conditions and that of stop codons at 37°C.

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We examined Hsp90 ts mutations for structural and physical patterns. We found that ts mutations tended to concentrate at certain amino acid positions of Hsp90 (Figure 4B). The clustering of ts mutations was significant compared to random simulations. Positions with greater than four ts mutations were spread across all three domains of Hsp90 (Figure 4C) with the largest cluster occurring in the C domain of Hsp90. The C domain forms a constitutive homodimer that is critical for function (Wayne and Bolon, 2007). Of note, homo-oligomerization domains may have a larger ts potential because all subunits contribute to folding and dimerization essentially multiplying the impacts of mutations (Lynch, 2013). To explore the physical underpinnings of ts mutations, we examined if they were buried in the structure or surface exposed. Mutations at buried residues tend to have a larger impact on protein folding energy compared to surface residues (Chakravarty and Varadarajan, 1999). Consistent with the idea that many ts mutations may disrupt protein folding at elevated temperature, substitutions that confer a ts phenotype are enriched in buried residues (Figure 4D). Also consistent with this idea, ts mutations tend to have negative Blosum scores (Henikoff and Henikoff, 1992; Figure 4E), a hallmark of disruptive amino acid changes.

Because growth at elevated temperatures requires higher levels of Hsp90 protein (Borkovich et al., 1989), some ts mutations are likely due to a reduced function that is enough for growth at standard temperature, but is insufficient at 37°C (Nathan and Lindquist, 1995). We reasoned that we could distinguish these mutants by examining how growth rate depended on the expression levels of Hsp90. We expect that destabilizing mutants that cause Hsp90 to unfold at elevated temperature would not support efficient growth at 37°C independent of expression levels. In contrast, we expect mutants that reduce Hsp90 function to exhibit an expression-dependent growth defect at 37°C. We tested a panel of ts mutations identified in the bulk competitions at high and low expression levels (Figure 4F). The dependence of growth rate at 37°C on expression level varied for different Hsp90 ts variants. The I66E, G170D and L499R Hsp90 mutants have no activity at 37°C irrespective of expression levels. These disruptive substitutions at buried positions likely destabilize the structure of Hsp90. In contrast, increasing the Hsp90 expression levels at least partially rescued the growth defect for five ts variants (L50D, K102A, D180L, K398L, K594I), indicating that these variants do not provide enough Hsp90 function for robust growth at elevated temperature. All five of these expression-dependent ts variants were located at surface positions. Thus, for the ts mutants we tested individually, we see a correlation between location of the mutation and type of ts mutation. Destabilizing mutations tend to be buried and mutants with reduced function tend to be surface exposed, indicating that the location of ts mutations can delineate these different mechanistic classes.

Hsp90 potential for adaptation to environmental stress

Numerous Hsp90 variants provided a growth benefit compared to the wild-type sequence in stress conditions. The largest number of beneficial variants in Hsp90 occurred in high temperature and diamide conditions (Figure 5A, Figure 5—source data 1). Multiple lines of evidence indicate that these mutants are truly beneficial variants and not simply measurement noise. First, the beneficial amino acids generally exhibited consistent selection coefficients among synonymous variants (Figure 5—figure supplement 1A). Second, beneficial mutants in diamide and high temperature tend to cluster at certain positions (Figure 5B), which would not be expected for noise. Finally, we confirmed the increased growth rate at elevated temperature of a panel of variants analyzed in isolation (Figure 5—figure supplement 1B). The fact that beneficial mutations in elevated temperature and diamide often clustered at specific positions in Hsp90 indicates that the wild-type amino acids at these positions are far from optimum for growth in these conditions. In contrast, the apparent beneficial mutations in other conditions did not tend to cluster at specific positions (Figure 5—figure supplement 2).

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List of all beneficial mutants and associated selection coefficients at 37°C and in diamide.

Beneficial mutants were defined as variants with selection coefficients two standard deviations greater than wild-type synonyms.

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To obtain a more general picture of the potential for adaptation derived from the full fitness distributions, we used Fisher’s Geometric model (FGM) (Fisher, 1930). According to FGM, populations evolve in an n-dimensional phenotypic space, through random single-step mutations, and any such mutation that brings the population closer to the optimum is considered beneficial. An intuitive hypothesis derived from FGM is that the potential for adaptation in a given environment (i.e. is the availability of beneficial mutations) depends on the distance to the optimum. In order to estimate the distance to the optimum d, we adopted the approach by Martin and Lenormand and fitted a displaced gamma distribution to the neutral and beneficial mutations for each environment (Martin and Lenormand, 2006). We observed that the yeast populations were furthest from the optimum in elevated temperature and diamide (d = 0.072 and 0.05, respectively), followed by nitrogen deprivation (d = 0.023), high salinity and ethanol (d = 0.021) and standard (d = 0.014). This suggests that exposure to elevated temperature and diamide results in the largest potential for adaptation and is consistent with the observation of the largest proportions of beneficial mutations in these environments. Interestingly, previous results from a 9-amino-acid region in Hsp90 indicated that there was very little potential for adaptation at high temperature (36°C) as compared with high salinity (Hietpas et al., 2013). This apparent contradiction between results from the full Hsp90 sequence and the 582–590 region indicates that a specific region of the protein may be already close to its functional optimum in a specific environment, whereas there is ample opportunity for adaptation when the whole protein sequence is considered.

In diamide and elevated temperature, the clustered beneficial positions were almost entirely located in the ATP-binding domain and the middle domain (Figure 5C), both of which make extensive contacts with clients and co-chaperones (Ali et al., 2006; Meyer et al., 2003; Meyer et al., 2004; Roe et al., 2004; Verba et al., 2016; Zhang et al., 2010). Beneficial mutations in elevated temperature and diamide conditions were preferentially located on the surface of Hsp90 (Figure 5D) at positions accessible to binding partners. Analyses of available Hsp90 complexes indicate that beneficial positions were disproportionately located at known interfaces with co-chaperones and clients (Figure 5E). Clustered beneficial mutations are consistent with disruptive mechanisms because a number of different amino acid changes can lead to disruptions, whereas a gain of function is usually mediated by specific amino acid changes. Amino acids that are beneficial in diamide and elevated temperature tend to exhibit deleterious effects in standard conditions (Figure 5F), consistent with a cost of adaptation. In comparison, wild-type-like mutations in diamide and high temperature tend to exhibit wild-type-like fitness in standard conditions (Figure 5—figure supplement 3). We conjecture that the clustered beneficial mutations are at positions that mediate the binding affinity of subsets of clients and co-chaperones and that disruptive mutations at these positions can lead to re-prioritization of multiple clients. The priority or efficiency of Hsp90 for sets of clients can in turn impact most aspects of physiology because Hsp90 clients include hundreds of kinases that influence virtually every aspect of cell biology.

In the first seven amino acids of Hsp90, we noted both a large variation in the selection coefficients of synonymous mutations at elevated temperature and that many nonsynonymous substitutions at these positions generated strong beneficial effects (Figure 5—figure supplement 4A,B). Synonymous mutations at these positions were only strongly beneficial at high temperature where Hsp90 protein levels are limiting for growth. Analysis of an individual clone confirms that a synonymous mutation at the beginning of Hsp90 that was beneficial at high temperature was expressed at higher level in our plasmid system (Figure 5—figure supplement 4C,D). These results are consistent with a large body of research showing that mRNA structure near the beginning of coding regions often impacts translation efficiency (Li, 2015; Plotkin and Kudla, 2011; Tuller et al., 2010), and that adaptations can be mediated by changes in expression levels (Lang and Desai, 2014).

Natural selection favors Hsp90 variants that are robust to environment

We next examined how experimental protein fitness maps compared with the diversity of Hsp90 sequences in current eukaryotes. We analyzed Hsp90 diversity in a set of 267 sequences from organisms that broadly span across eukaryotes. We identified 1750 amino acid differences in total that were located at 499 positions in Hsp90. We examined the experimental growth effects of the subset of amino acids that were observed in nature. While the overall distribution of selection coefficients in all conditions was bimodal with peaks around neutral (s = 0) and null (s = −1), the natural amino acids were unimodal with a peak centered near neutral (Figure 6A, Figure 6—source data 1). The vast majority of natural amino acids had wild-type-like fitness in all conditions studied here (Figure 6B and C). Whereas naturally occurring amino acids in Hsp90 were rarely deleterious in any experimental condition, they were similarly likely to provide a growth benefit compared to all possible amino acids (5%). This observation indicates that condition-dependent fitness benefits are not a major determinant of natural variation in Hsp90 sequences. Instead, our results indicate that natural selection has favored Hsp90 substitutions that are robust to multiple stressful conditions (Figure 6D). Beneficial mutations in heat and diamide indicate that there is room for improvement in Hsp90 function in individual conditions. The clustering of beneficial mutations at known binding interfaces suggests that the optimal binding affinity for partner proteins may depend on growth conditions. We propose that natural variants of Hsp90 have been selected for binding properties that are robust to different stresses rather than specific to individual conditions.

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List of selection coefficients of all natural variants of Hsp90 in all environmental conditions.

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Epistasis may provide a compelling explanation for the naturally occurring amino acids that we observed with deleterious selection coefficients. Analyses of Hsp90 mutations in the context of likely ancestral states has demonstrated a few instances of historical substitutions with fitness effects that depend strongly on the Hsp90 sequence background (Starr et al., 2018). Indeed, many of the natural amino acids previously identified with strong epistasis (E7A, V23F, T13N) are in the small set of natural amino acids with deleterious effects in at least one condition. Further analyses of natural variants under diverse environmental conditions will likely provide insights into historical epistasis and will be the focus of future research.

In this study, we analyzed the protein-wide distribution of fitness effects of Hsp90 across standard and five stress conditions. We found that environment has a profound effect on the fates of Hsp90 mutations. Each environmental stress varies in the strength of selection on Hsp90 mutations; heat and diamide increase the strength of selection and ethanol and salt decrease the strength of selection. While proximity to ATP is the dominant functional constraint in standard conditions, the influence of client and co-chaperone interactions on growth rate dramatically increases under stress conditions. Additionally, beneficial mutations cluster at positions that mediate binding to clients and cochaperones. The fact that different Hsp90 binding partners have distinct environmental dependencies suggests that Hsp90 can reprioritize clients that in turn impacts many downstream signaling pathways.

Our results demonstrate that mutations to Hsp90 can have environment-dependent effects that are similar to the stress-induced changes to the function of wild-type Hsp90 that have been shown to contribute to new phenotypes (Jarosz et al., 2010). The low frequency of environment-dependent amino acids in Hsp90 from extant eukaryotes indicates that this type of evolutionary mechanism is rare relative to drift and other mechanisms shaping Hsp90 sequence diversity.

We observed distinct structural trends for mutations that provide environment-dependent costs and benefits. Many mutations in Hsp90 caused growth defects at elevated temperature where Hsp90 function is limiting for growth. These temperature-sensitive mutations tended to be buried and in the homodimerization domain, consistent with an increased requirement for folding stability at elevated temperatures. In contrast, beneficial mutations tended to be on the surface of Hsp90 and at contact sites with binding partners, suggesting that change-of-function mutations may be predominantly governed by alterations to binding interactions. Mutations that disrupt binding to certain clients can lead to the re-prioritization of others, which, due to Hsp90’s central role in numerous cellular pathways, has the potential to modify integral networks in response to stress. Once more comprehensive data is available on Hsp90-client binding sites, it may be possible to simulate this rewiring of cellular networks, providing insight into the causes of the beneficial mutations. However, presently, the large number of clients with unknown binding sites makes these analyses challenging. In the future, comparing Hsp90 client-interactomes (Taipale et al., 2012) may help delineate adaptive biochemical mechanisms.

Limitations:

Our experimental setup has limitations that we have tried to account for in our analyses and conclusions. For example, we measured the fitness effects of Hsp90 under artificially low expression where yeast growth rates are tightly coupled to function of Hsp90 in order to provide a sensitive readout of fitness defects (Jiang et al., 2013). Expression of Hsp90 under this promoter remains stable in the stresses tested (Figure 6—figure supplement 1). However, this defined promoter does not capture the native transcriptional regulation and may not fully recapitulate translational and post-translational regulation controlling hsp82. While these levels of regulation of Hsp90 are clearly important physiologically, the sensitive readouts of fitness that we measured appear to capture critical features of Hsp90 with regard to biochemical function and evolutionary mechanism. For example, virtually all deleterious mutants measured in this study under stress conditions appear to have also been subject to purifying selection in nature.

In addition, the experimental strain used in this study is deleted for the constitutively expressed paralog of hsp82, hsc82. Hsp82 and hsc82 are functionally overlapping, essential genes with 97% sequence identify (16 amino acid differences) that can compensate for each other’s loss-of-function in normal growth conditions (Girstmair et al., 2019). The high sequence identity between the two paralogs indicates that they are both under similar selection pressure. Despite the high sequence identity, a number of distinct differences have been noted in stability, conformational cycles, and client interactomes. Experimental evidence indicates that hsp82 is more stress-specific, and more stable to unfolding (Girstmair et al., 2019). Further efforts will be required to resolve how distinctions between Hsp90 paralogs contribute to function and selection.

Relationship to prior work:

A handful of studies have assessed the impact of environment on the fitness landscape of genes (Dandage et al., 2018; Hietpas et al., 2013; Li and Zhang, 2018; Mavor et al., 2016). For example, Dandage, Chakraborty and colleges investigated the effects of temperature and chemical chaperones on the fitness landscape of the Gentamicin resistance gene and found that protein stability and distance to the ligand binding site are the molecular properties with the strongest correlations with fitness (Dandage et al., 2018). To understand the strength of the molecular constraints on Hsp90 on a whole protein level, we performed similar analyses (Figure 6—figure supplement 2). Consistent with the Gentamicin study, we find the features that best correlate with fitness are protein stability and distance from the active site. The constraint of protein stability is the highest at 37°C, indicating increased dependence on stability at the higher temperature. In addition, distance from the ATP-binding site imposes strong molecular constraints on Hsp90, signifying the importance of ATP hydrolysis on Hsp90 function. While individual features correlate with fitness effects and show environmental dependence, single features are unable to capture the majority of observed variance in fitness effects, consistent with a complex set of physical properties that underlie fitness effects in both proteins.

In another study of the effect of environment on mutational fitness, Li and Zhang detected pervasive genotype-by-environment interactions between a yeast tRNA gene and environment (Li and Zhang, 2018). They found that the correlation of the fitness between mutations in each tested environment was linear such that the fitness landscape in one environment together with a change in slope could be used to accurately predict fitness effects in the second environment. In this study, we observed a large impact of environment on Hsp90 fitness; however, we observe many fitness effects that deviate from a linear relationship between environments. While linear models can predict the fitness of some mutations in different environments, it would not predict many of the types of mutations that are focuses of this study, such as mutations that exhibit an adaptive trade-off, those with beneficial effects in one environment that become deleterious in another. In addition, the linear model would not predict the large group of ts mutations with wild-type fitness in standard conditions and null fitness at 37°C. As environmental-dependent protein fitness landscapes are analyzed for an increasing set of genes, it will provide opportunities to explore how different protein properties such as the number of binding partners may contribute to global trends.

Importantly, our results demonstrate that while mutations to Hsp90 can provide a growth advantage in specific environmental conditions, naturally occurring amino acids in Hsp90 tend to support robust growth over multiple stress conditions. The finding of beneficial mutations in Hsp90 in specific conditions suggests that similar long-term stresses in nature can lead to positive selection on Hsp90. Consistent with previous work (Hietpas et al., 2013), we found that experimentally beneficial mutations tended to have a fitness cost in alternate conditions (Figure 5F). This indicates that natural environments which fluctuate among different stresses would reduce or eliminate positive selection on Hsp90. Therefore, our results suggest that natural selection on Hsp90 sequence has predominantly been governed by strong purifying selection integrated over multiple stressful conditions. Taken together, these results support the hypothesis that natural populations might experience a so-called ‘micro-evolutionary fitness seascape’ (Mustonen and Lässig, 2009), in which rapidly fluctuating environments result in a distribution of quasi-neutral substitutions over evolutionary time scales.

Next generation sequencing data has been deposited to the NCBI short read archive (Project # PRJNA593726). Tabulated raw counts of all variants in all conditions are included in the manuscript in Figure 1—source data 1 and Figure 2—source data 2. Source data files have been provided for Figure 1, 2, 3, 4, 5 and 6.

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Author details

1. Julia M Flynn

   Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5490-393X

2. Ammeret Rossouw

   Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Data curation, Software, Investigation, Methodology

   Competing interests

   No competing interests declared

3. Pamela Cote-Hammarlof

   Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Conceptualization, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

4. Inês Fragata

   Instituto Gulbenkian de Ciência, Oeiras, Portugal

   Contribution

   Data curation, Software, Formal analysis, Validation, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6865-1510

5. David Mavor

   Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Data curation, Software, Formal analysis

   Competing interests

   No competing interests declared

6. Carl Hollins III

   Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Formal analysis, Validation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0410-9639

7. Claudia Bank

   Instituto Gulbenkian de Ciência, Oeiras, Portugal

   Contribution

   Data curation, Software, Formal analysis, Funding acquisition, Validation, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4730-758X

8. Daniel NA Bolon

   Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   Dan.Bolon@umassmed.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5857-6676

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