Author response: Super Spy variants implicate flexibility in chaperone action

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. https://doi.org/10.7554/eLife.01584.001 eLife digest Proteins are made from long chains of smaller molecules, called amino acids, that twist and fold into complex three-dimensional shapes. Folding into the correct shape is crucial for a protein to function properly because many proteins work by binding to certain other proteins or molecules, like a key fitting into a lock. Additional proteins called chaperones often help with this folding process, and it has been proposed that chaperones must be particularly flexible in order to cope with the changes in the shape of the different proteins being folded. However, studying this hypothesis directly has proven to be difficult. Now, Quan et al. have tackled this challenge by using a bacterial assay—that they had developed previously—and which links the correct folding of a test protein to cell survival and growth in the presence of an antibiotic. This approach was formerly used to identify a new chaperone called Spy, and Quan et al. have now used it to find variants of this protein that perform as even better chaperones. This assay identified several variants of Spy that could stabilise an unstable test protein even more effectively than the wild-type Spy can. All of these variants were also better than the wild-type Spy at stabilising two other unfolded proteins—and so were dubbed ‘super Spy’ proteins. The mutations in the super Spy variants altered a region on the surface of Spy, which additional experiments revealed was likely to be involved in binding to the partner proteins. Furthermore, prior to binding to these partner proteins, the super Spy variants appear more flexible than the wild-type Spy protein. Quan et al. suggest that this increase in flexibility allows the super Spy variants to bind more tightly to a range of substrates, thus optimising their chaperone function. https://doi.org/10.7554/eLife.01584.002 Introduction Despite years of intense effort, the precise mechanism by which chaperones interact with proteins to enhance their folding is not entirely clear. We reasoned that we might gain insight into this long-standing problem by isolating and characterizing chaperone variants that exhibit improved chaperone activity. A genetic selection that we had developed previously gave us a unique opportunity to pursue these aims. This selection uses a folding biosensor to directly link protein stability to antibiotic resistance. The biosensor consists of an unstable protein inserted into β-lactamase, a selectable marker that encodes penicillin resistance (Foit et al., 2009). Stabilization of the unstable protein results in higher levels of antibiotic resistance. We showed that the stabilization could be due to mutations within the unstable protein itself (Foit et al., 2009), addition of chemical chaperones to the growth media (Hailu et al., 2013), or host variants that stabilize the unstable protein (Quan et al., 2011). We isolated host variants that greatly stabilize poorly folded variants of immunity protein 7 (Im7), increasing their steady-state concentrations in the cell. We found that this stabilization occurs through the induction of a previously uncharacterized chaperone called Spy (Quan et al., 2011). We obtained evidence that Spy acts in an ATP-independent manner to help protect bacterial cells from a number of conditions that lead to widespread protein denaturation and aggregation, such as treatment with tannin, ethanol, or butanol (Quan et al., 2011). The crystal structure of Spy shows that it forms an unusual cradle shaped dimer (Figure 1; Quan et al., 2011; Kwon et al., 2010). When we attached environmentally sensitive probes to various sites in Spy, including the concave and convex surfaces, nearly all showed substantial changes in fluorescence upon interaction with the client protein casein. These results suggest that client binding may occur over large regions of Spy, that Spy might undergo significant conformational changes upon client binding, or a combination of both (Quan et al., 2011). Figure 1 with 2 supplements see all Download asset Open asset Surface presentations of the crystal structure of Spy (PDB ID: 3O39). The majority of activity-enhancing mutations localize to areas adjacent to hydrophobic patches. (A) Surface properties of Spy. Backbone atoms are shown in white, hydrophobic side chain atoms in yellow, and polar and charged side chain atoms in blue. Black dashed lines circle the two predominant hydrophobic patches P1 and P2. (B) Sites accommodating beneficial mutations. Side chain atoms of the residues identified as mutations in the genetic selection are shown in red. Q25 is in the disordered N-terminus, which is not visible in the crystal structure. Q49L, H96L, and Q100L would expand the total hydrophobic area of P1 and P2. https://doi.org/10.7554/eLife.01584.003 We decided to investigate the mechanism by which Spy interacts with proteins and learn more about Spy’s properties as a chaperone. We used a genetic selection similar to that used to discover Spy in an attempt to further enhance Spy’s chaperone properties. We have now isolated Spy variants with improved ability to stabilize a poorly folded client protein (Im7 L53A I54A) in vivo. These variants also showed improved ability to prevent the aggregation of client proteins in vitro. Many of these Spy variants contain residue substitutions that act to expand a hydrophobic region present on the protein’s concave surface. Crosslinking and hydrogen-deuterium exchange measurements suggest that this hydrophobic region is involved in client protein interaction. Our optimized Spy variants bind the client protein Im7 more tightly than wild type Spy does but are generally less stable suggesting that flexibility is important in the function of Spy as a chaperone. Results Identification of Spy variants with improved ability to stabilize a poorly folded client protein We expressed a protein stability biosensor in an Escherichia coli strain that co-expresses the gene for the chaperone Spy under the IPTG inducible Trc promoter. The stability biosensor consists of a tripartite fusion that contains the unstable protein Im7 L53A I54A inserted into β-lactamase under the constitutive β-lactamase promoter (Foit et al., 2009). This partially unfolded variant of Im7 was chosen because Spy overproduction is known to stabilize it in vivo (Quan et al., 2011). Increasing the expression level of the chaperone Spy by increasing IPTG concentrations results in improved penicillin resistance encoded by the β-lactamase-Im7 L53A I54A biosensor (Figure 1—figure supplement 1A,B, focus on wild-type [WT] traces [black lines]). We reasoned that if mutations in Spy increase its specific activity as a chaperone, they should also be capable of enhancing the stability of the biosensor and thereby also enhance antibiotic resistance. Our ability to link protein folding to antibiotic resistance gives us a unique opportunity to select for activity-enhancing mutations in a chaperone. Analysis of the reasons behind the improved chaperone ability of activity enhancing mutants of Spy should inform us about Spy’s catalytic mechanism and perhaps also tell us what makes for a good chaperone. We reasoned that activity-enhancing mutations would be more informative in general than those that decreased function, in part because there are a wider variety of uninteresting reasons that mutations can disrupt function such as those causing chain termination. If we succeeded at all in getting activity enhancing mutations we anticipated obtaining two types of mutations. We might obtain those that acted in a substrate specific manner that improved the action of Spy only against the substrate for which they were selected on, and variants that generally improved the activity of Spy against multiple substrates. If we succeeded in obtaining this latter type of mutations, they should be particularly informative as to what makes a protein an effective chaperone. To obtain activity-enhanced Spy variants, we used an error-prone PCR-based approach (McCullum et al., 2010) that targeted the mature protein encoding region of the spy gene on pCDFTrc-Spy to create a plasmid library of ∼106 members that contained an average of 1.2 nucleotide mutations per spy gene. This variant library was transformed into SQ2041, a spy null strain of E. coli that contains the stability biosensor (see strain list in Table 1). We plated the mutant library onto LB plates that contained 0.1 mM IPTG (to induce Spy) and 4 mg/ml penicillin, the concentration at which a strain co-expressing wild-type Spy and the biosensor (strain SQ2068) fails to grow. Using this selection approach, we isolated 65 Spy variants that, when co-expressed with the biosensor, showed improved antibiotic resistance compared to cells that co-express wild-type Spy. Table 1 Strain list https://doi.org/10.7554/eLife.01584.006 StrainGenotype or relevant characteristicsSourceSQ765MG1655 (F¯ λ¯ ilvG¯ rfb-50 rph-1), ΔhsdR(Quan et al., 2011)SQ2041SQ765, ΔampC, Δspy, pBR322 bla::GSlinker Im7 L53A I54A (Foit et al., 2009)This studySQ2068SQ2041, pCDFTrc-SpyThis studyLW53SQ2041, pCDFTrc-Spy Q100LThis studyLW54SQ2041, pCDFTrc-Spy L32PThis studyLW55SQ2041, pCDFTrc-Spy F115IThis studyLW56SQ2041, pCDFTrc-Spy Q49LThis studyLW57SQ2041, pCDFTrc-Spy F115LThis studyLW58SQ2041, pCDFTrc-Spy H96LThis studyLW59SQ2041, pCDFTrc-Spy Q25RThis study Remarkably, 48 (74%) of the isolated Spy variants contained a glutamine to leucine mutation at amino acid 100. For 20 of these variants, this alteration (Q100L) was the only mutation present, and strains expressing a Spy Q100L variant emerged from at least four independent mutagenesis and selection experiments. Other single mutations that answered the selection included Q25R, L32P, and F115I. There were also a number of other mutations (Q49L, H96L, and F115L) that were found independently 2–3 times in combination with other amino acid substitutions. To verify that these Spy mutations enhance the antibiotic resistance of the co-expressed biosensor when they are present as single mutations, we introduced the individual mutations Q25R, L32P, Q49L, H96L, Q100L, F115I, and F115L into the spy gene on the plasmid pCDFTrc-spy by site-directed mutagenesis and transformed the resulting plasmids into SQ2041, the spy knockout strain co-expressing the biosensor. All of these strains except the one containing Q49L showed improved penicillin resistance compared to strains expressing wild-type Spy at a wide range of IPTG concentrations (Figure 1—figure supplement 1A); the relative minimal inhibitory concentrations (MICs) were up to twofold higher than the MIC of cells co-expressing wild-type Spy. Improved antibiotic resistance of Spy variants is due to enhanced chaperone activity One simple explanation for the increased penicillin resistance observed in the mutated strains might be increased Spy levels. Such an increase could occur through translational or posttranslational effects such as an increase in Spy stability. To examine these possibilities, we measured the steady-state expression levels of Spy in these strains when induced by different IPTG concentrations. All variants exhibited Spy levels that were within 20% of wild-type except Q49L, which showed a Spy level that was half that of wild-type (Figure 1—figure supplement 1B). These results suggest that the observed increases in MIC (up to twofold) for the variant strains are not simply due to increased expression levels of the chaperone. We then measured the specific in vivo activity of our Spy variants by normalizing the maximal MIC values for penicillin V of the variant strains to the amount of Spy variant proteins found in these strains (Figure 1—figure supplement 1, ‘Materials and methods’). All strains co-expressing the selected Spy variants (including Q49L) had normalized MICs 1.4–2.2-fold higher than SQ2068 (Table 2), indicating that the variant Spy proteins have higher specific activity than wild-type Spy. Furthermore, quantitative western blots showed that the increased MICs are linearly correlated with the steady-state levels of the folding biosensor (Figure 1—figure supplement 1C). Table 2 Properties of Spy variants https://doi.org/10.7554/eLife.01584.007 Spy variantsMICnormActivity (aldolase agg. Prev)Activity (aldolase refold)Activity (α-LA agg. Prev)kon (× 105 mol−1 s−1)koff (s−1)KD (µM)Tm (°C)ΔHm (Kcal mol−1)ΔCp (Kcal K−1 mol−1)ΔGNU (25°C) (Kcal mol−1)WT11113.98 ± 0.110.456 ± 0.0111.15 ± 0.02748.1 ± 0.166.6 ± 1.50.644.24 ± 0.10Q25R1.446.90 ± 0.641.38 ± 0.312.44 ± 0.892.29 ± 0.130.198 ± 0.0050.87 ± 0.03446.3 ± 0.473.7 ± 0.80.984.20 ± 0.10L32P1.922.52 ± 0.114.85 ± 0.572.10 ± 0.741.51 ± 0.140.030 ± 0.0020.20 ± 0.00331.0 ± 0.252.1 ± 2.50.710.99 ± 0.02Q49L1.602.88 ± 0.144.25 ± 0.661.93 ± 0.662.30 ± 0.060.176 ± 0.0090.76 ± 0.01852.0 ± 0.259.9 ± 1.00.684.19 ± 0.10H96L1.622.02 ± 0.321.90 ± 0.321.64 ± 0.492.68 ± 0.140.266 ± 0.0101.00 ± 0.02250.1 ± 0.156.2 ± 3.00.713.66 ± 0.23Q100L2.191.34 ± 0.054.20 ± 0.442.12 ± 0.751.19 ± 0.130.027 ± 0.0020.23 ± 0.01353.8 ± 0.628.9 ± 1.10.232.24 ± 0.11F115L1.521.98 ± 0.324.85 ± 0.562.30 ± 0.832.73 ± 0.010.245 ± 0.0070.90 ± 0.02841.3 ± 0.256.6 ± 4.90.762.60 ± 0.22F115I1.652.21 ± 0.124.33 ± 0.492.34 ± 0.852.82 ± 0.150.328 ± 0.0171.17 ± 0.09741.7 ± 0.454.3 ± 1.50.982.43 ± 0.11 ΔGNU(25°C) is the free energy of stabilization at 25°C (NU dictates the transition from folded state to unfolded state), ΔHm is the change in enthalpy at Tm which is the melting temperature and ΔCp is the change in heat capacity associated with the unfolding of the Spy variant. agg. prev: aggregation prevention. Fold activity expresses relative to WT. Values after the ± sign are standard errors. MICnorm is measured for cells (SQ2068, LW53-59) expressing the pBR322 bla::GSlinker Im7 L53A I54A plasmid and various Spy constructs. kon, koff, and KD are kinetic parameters describing the interaction between Im7 L53A I54A and the Spy variants. To test whether the Spy variants’ increased chaperone activity was general or client specific, we purified the variant proteins and tested their chaperone activity in vitro using two standard chaperone clients: reduced denatured α-lactalbumin (α-LA) and chemically denatured aldolase. In genetic selections one usually gets what you select for, thus we had anticipated that the variants we obtained would show an improved ability to refold Im7. These mutations, would at a minimum, likely to be informative about the factors involved in Spy-Im7 interactions. We however considered it unlikely that they would show generally improved chaperone activity for at least two reasons. First, in a wide variety of laboratory evolution experiments where variant enzymes are selected that show improved activity against one substrate, often though not always show decreased activity against other unrelated substrates (Goldsmith et al., 2012; Yang et al., 2013). More specifically, other efforts at improving chaperone activity, though showing some success in generating mutants that were better with the substrates they were selected on, in general showed decreased chaperone activity against other substrates (Wang et al., 2002; Aponte et al., 2010; Schweizer et al., 2011). For instance Wang et al, through the use of a multistep screening process, succeeded in isolating GroEL variants that enhanced the expression of GFP and circularly permuted versions of GFP 3-8-fold, presumably by enhancing folding of GFP in vivo. However these variants were defective in all other measures of GroEL function tested including ability to support bacterial growth at high temperature, in vivo folding of the GroEL substrate HrcA, and phage lambda and Mu growth (whose growth dependency on GroEL and GroES historically led to the naming of the GroE genes [Georgopoulos et al., 1972]). These GroEL variants in vitro were also no better than wild type GroEL in enhancing the yield of active GFP. The authors concluded that increased GFP folding of these variants ‘comes at the expense of the ability of GroEL/S to fold its natural substrates’ (Wang et al., 2002). The majority of the DnaK variants Aponte et al isolated based on an improved ability to fold an unstable variant of chloramphenicol acetyl transferase in vivo turned out to be inferior to wild type DnaK in refolding luciferase in vitro, though it needs to be mentioned that they did succeed in isolating four variants that showed a slightly improved in vitro refolding yield for luciferase ranging from 1.2 to 1.9-fold (Aponte et al., 2010). Given the apparent difficulty in isolating chaperone or enzyme variants that show generally enhanced activity against a variety of substrates, we were surprised that all 7 Spy variants that we had isolated based on their ability to fold Im7 in vivo were significantly more active in preventing aggregation of both chemically denatured α-lactalbumin and aldolase in in vitro assays. In the aldolase aggregation assay, they were 1.3–6.9-fold more active than wild type, and in the α-lactalbumin aggregation assay, they were 1.6–2.4-fold more active (Table 2). We also tested for the activity of these chaperone variants in their ability to facilitate aldolase refolding. Six of the seven of the variants were found to be more active than is wild type Spy in the range of 1.9–4.9-fold. The one exception, Q25R, was measured to marginally increase refolding yield (1.4-fold) (See Table 2). Because our Spy variants showed improved chaperone activity towards at least three client proteins (Im7, aldolase and α-lactalbumin), we called them ‘super-Spy’ variants. Hydrophobic areas may be involved in client binding of activity-enhanced Spy variants We mapped the activity-enhancing mutations identified in the selection onto Spy’s crystal structure and found that many of them were located close to each other. Most of them mapped immediately adjacent to the two predominant hydrophobic patches on the concave surface (P1, P2) of the cradle interior (Figure 1A,B)—the region that we had previously hypothesized might be involved in client binding (Quan et al., 2011). The most commonly observed substitutions (Q100L, which occurred in 74% of the variants, and H96L and the Q49L, which occurred in ∼5% and ∼3% of the variants, respectively) change polar or charged residues (glutamine and histidine) into the hydrophobic amino acid leucine, thereby increasing the area of the hydrophobic region. One proposed mechanism for chaperone function is via blocking hydrophobic regions present on client proteins, thereby preventing their aggregation (Hartl et al., 2011). Simple expansion of peptide-binding hydrophobic regions on our Spy variants could thus be one straightforward way to explain their improved chaperone activity. There are some expectations of this simple model:(1) chaperone variants with a larger or stronger hydrophobic patch will have enhanced affinity for client proteins, and (2) client proteins are likely to interact with regions on the 3D structure of the chaperone that are adjacent to the sites mutated in our selected Spy variants. Alternatively, the Spy mutations could increase chaperone efficacy in other less direct ways. For example, they could map to sites distant from the active site of the chaperone and exert their beneficial action through allosteric effects. To help distinguish between these possible models and to better understand how Spy interacts with its clients, we decided to map the site(s) with which Spy binds the client protein Im7. To achieve this, we:(1) examined the effects of Im7 binding on hydrogen-deuterium exchange in Spy, (2) investigated the proteolytic sensitivity of the chaperone in the presence and absence of Im7, and (3) crosslinked Spy to its client. Hydrogen-deuterium exchange identifies Spy residues involved in client binding Hydrogen-deuterium exchange at individual peptide bond amides is determined by the protection of amides from due to of structure or client To the effects of client protein binding on we compared the level of hydrogen-deuterium exchange of free Spy with that of a Spy-Im7 complex using a We Spy with a acid peptide from Im7 L53A I54A which binds Spy with a KD (see ‘Materials and for of peptide and Using exchange we obtained evidence that Spy amides more upon Spy-Im7 complex compared to free Spy (Figure supplement changes in protection were mapped to the residues located in and regions of Spy (Figure supplement 1B). We were to localize the improved Spy protection in the presence of client to several specific and The protection not only the flexible and and which are not present in the crystal but into the and as (Figure 2). protection from hydrogen-deuterium exchange in these regions could of these Spy residues in the interaction with Im7 or could the folding of these flexible regions upon client binding, or a combination of Figure 2 with 2 supplements see all Download asset Open asset exchange and on Spy for Im7 L53A I54A or Im7 peptide atoms on the bond that are upon addition of Im7 are shown as of Spy from upon addition of Im7 L53A I54A are shown as and with atoms and Im7 binding sites in Spy a approach to the Im7 binding site in Spy, we used a assay with a to the and regions in Spy and after Im7 binding as by their to the sites in Spy that show altered may be directly involved in Im7 binding or be the Im7 binding that it is also possible that Im7 binding a significant conformational change or change in flexibility in Spy that the of certain In the absence of Im7, the flexible and to a the flexible of Spy are more to than the regions (Figure supplement 2). In the presence of Im7, a number of sites including and show significant protection compared to free Spy (Figure 2). changes in occur on both the and of the cradle due to the of the Spy These changes suggest that client binding occurs over large of Spy or that client binding conformational changes or changes in or a combination of these these with the exchange results that Im7 peptide binding a relative large area on the Spy the regions and the of the and the of the of the and the of the of the Spy cradle (Figure 2). Crosslinking key residues directly involved in interactions with client To further map the of the site on Spy, we Crosslinking information about the between two residues as determined by the of the in the Identification of the crosslinked sites on a protein complex thus information and for the two amino acid residues that are We experiments on Spy and the peptide of residues from Im7. Crosslinking was using our developed et al., and the newly developed an 7 Given the long of the and the flexibility of the crosslinked side we were not surprised to find multiple (Figure Table The most crosslinked residues are and on the of Spy. and in this region are also in peptide binding through changes in suggesting that this flexible of Spy might be involved in client interaction. Figure with 1 supplement see all Download asset Open asset residues mapped onto the crystal structure of Spy. Spy residues that were found crosslinked to Im7 peptide are shown in Crosslinking with a between the of Im7 and Spy these Spy and that can be crosslinked with also Spy to Im7 Spy to Im7 and Spy to Im7 Using we identified between the of Spy and of Im7. using and identified between Spy and Im7 and Spy and Im7 A of all identified is in Table Table Spy-Im7 for and amino acid residues of the crosslinked crosslinked residue within enzyme The crosslinked residues are and in the The residues shown and after the are the and residues of the peptide are shown to digest The was introduced from the fusion which were used for Spy or Im7 (Quan et al., 2011). The is used to this The the the more precise and the information that can To obtain

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