Báo cáo khoa học: Directed evolution of a histone acetyltransferase – enhancing thermostability, whilst maintaining catalytic activity and substrate specificity doc

Directed evolution of a histone acetyltransferase – enhancing thermostability, whilst maintaining catalytic activity and substrate specificity Hans Leemhuis 1 , Karl P. Nightingale 1,2 and Florian Hollfelder 1 1 Department of Biochemistry, University of Cambridge, UK 2 Chromatin and Gene Expression Group, Medical School, University of Birmingham, UK The post-translational modification of the histone N-terminal ‘tails’ plays a central role in the epigenetic regulation of gene expression [1]. These processes are highly integrated into transcriptional control mecha- nisms, with many histone modifying enzymes being associated with core components of the transcriptional machinery. A diverse group of these enzymes generate various marks in chromatin by the covalent modifica- tion of the histone tails (i.e. phosphorylation, methyla- tion, acetylation, etc.). The histone-code hypothesis [2,3] suggests that these marks define the functional status of the underlying DNA, leading to transcrip- tional activation or silencing via the recruitment of specific effector proteins. Histone acetylation is the process of acetylation of specific lysine residues in histones (Fig. 1) by a range of histone acetyltransferase (HAT; EC 2.3.1.48) enzymes and typically leads to gene activation. Histone acetyla- tion exerts functional effects via two mechanisms. First, it is associated with the charge neutralization of lysine residues, thereby reducing the interaction of the lysine- rich (and thus positively charged) histone tails with Keywords acetylation; chromatin; enzymology; epigentics; protein engineering Correspondence F. Hollfelder, Department of Biochemistry, 80 Tennis Court Road, University of Cambridge, Cambridge CB2 1GA, UK Fax: +44 1223 766002 Tel: +44 1223 766048 E-mail: fh111@cam.ac.uk Website: http://www.bioc.cam.ac.uk/uto/ hollfelder.html (Received 1 August 2008, revised 5 September 2008, accepted 17 September 2008) doi:10.1111/j.1742-4658.2008.06689.x Histone acetylation plays an integral role in the epigenetic regulation of gene expression. Transcriptional activity reflects the recruitment of oppo- sing classes of enzymes to promoter elements; histone acetyltransferases (EC 2.3.1.48) that deposit acetyl marks at a subset of histone residues and histone deacetylases that remove them. Many histone acetyltransferases are difficult to study in solution because of their limited stability once purified. We have developed a directed evolution protocol that allows the screening of hundreds of histone acetyltransferase mutants for histone acetylating activity, and used this to enhance the thermostability of the human P ⁄ CAF histone acetyltransferase. Two rounds of directed evolution significantly stabilized the enzyme without lowering the catalytic efficiency and substrate specificity of the enzyme. Twenty-four variants with higher thermostability were identified. Detailed analysis revealed twelve single amino acid mutants that were found to possess a higher thermostability. The residues affected are scattered over the entire protein structure, and are different from muta- tions predicted by sequence alignment approaches, suggesting that sequence comparison and directed evolution methods are complementary strategies in engineering increased protein thermostability. The stabilizing mutations are predominately located at surface of the enzyme, suggesting that the protein’s surface is important for stability. The directed evolution approach described in the present study is easily adapted to other histone modifying enzymes, requiring only appropriate peptide substrates and antibodies, which are available from commercial suppliers. Abbreviations DSC, differential scanning calorimetry; HAT, histone acetyltransferase; HDAC, histone deacetylase. FEBS Journal 275 (2008) 5635–5647 ª 2008 The Authors Journal compilation ª 2008 FEBS 5635 DNA. The subsequent decreased compaction of chro- matin facilitates access of DNA binding proteins (i.e. transcription factors). Second, acetylated lysine residues are recognized by ‘bromodomains’, a specific protein fold found in many transcriptional regulators and chromatin remodellers, suggesting that these proteins are recruited to regions of acetylated chromatin, and subsequently contribute to gene activation. HAT and histone deacetylase (HDAC) activities are typically found in multi-subunit complexes, which are recruited to their target loci by interactions with tran- scriptional activators, or repressors, respectively [4]. Several classes of histone modification enzymes, including HATs [5,6] and histone demethylases, are less active when studied in vitro (i.e. overexpressed as an individual polypeptide in the absence of interaction partners). A limitation of the biochemical characteriza- tion of the HAT enzymes is their low activity and stability in vitro. It is desirable to improve such mar- ginally stable proteins in order to be able to use and study them in biochemical experiments. Proteins from thermophiles [7] have adapted various strategies [8] to make them more thermostable, includ- ing the incorporation of stabilizing structural features, such as an increase in the number of charged residues and ion pairs [9,10], increased a-helical content [11], increased structural compactness [12] and entropic sta- bilization due to an increased lysine to arginine ratio [13]. Improved stability of proteins can be achieved by design and library-based methods. Site-directed muta- genesis based on sequence alignments and comparison of 3D structures has been successful in creating pro- teins with higher thermostability [14–19], although many designed mutations had no stabilizing effect at all. This emphasizes that our current ability to inte- grate the lessons from naturally thermostable proteins into engineering stable new structures is still far from perfect [20] and has ensured that directed evolution is increasingly being used to enhance the thermostability of proteins. This approach involves the generation of genetic diversity in the gene encoding the protein of interest, followed by screening for mutant proteins with the desired properties, and has been successfully applied to change or improve enzyme function (sub- strate selectivity and activity) and expression or to enhance the stability of proteins [21–28]. The general picture emerging from these studies is that just one or a few amino acid substitutions can be sufficient to increase the thermostability of a protein by up to tens of degrees Celsius and that it is generally hard to pre- dict which mutations will be stabilizing. In the present study, we describe a procedure that uses random mutagenesis and subsequent screening to identify substantially more thermostable mutants of the catalytic domain of the human HAT P ⁄ CAF, with- out affecting its catalytic activity or substrate specific- ity. P ⁄ CAF, p300 ⁄ CBP-associating factor, is a trans criptional coactivator with a variable N-terminal, a central HAT domain and a C-terminal bromodomain. Several stabilizing mutations at multiple residues throughout the protein were found to generate a more thermostable HAT. Results Generating P ⁄ CAF mutants with increased thermostability One thousand variants of the catalytic domain of P ⁄ CAF generated by error-prone PCR mutagenesis were screened for HAT activity following a mild heat challenge (22 °C for 2 h), as shown in Fig. 2. This Fig. 1. The acetylation of a lysine residue catalysed by P ⁄ CAF using acetyl-CoA as the acetyl donor. A B Fig. 2. Screening procedure used to generate thermostable P ⁄ CAF variants. (A) Acetylation of lysine residues is detected by an ELISA protocol using an antibody specific for acetylated lysine residues. The signal is amplified by a secondary antibody conjugated to horseradish peroxidase. (B) Overview of the selection procedure. Step 1: microtitre plates with liquid medium (200 lL) were inocu- lated with single transformants and grown overnight. Step 2: 25 lL of culture was transferred to a second plate containing fresh med- ium with isopropyl thio-b- D-galactoside to induce protein expres- sion. Step 3: cells were harvested by centrifugation and lysed with BugBuster. Following a heat challenge, the lysates were directly used to detect histone acetyltransferase activity, as shown in (A). Stabilizing a human histone acetyltransferase H. Leemhuis et al. 5636 FEBS Journal 275 (2008) 5635–5647 ª 2008 The Authors Journal compilation ª 2008 FEBS procedure yielded 24 P ⁄ CAF mutants with an increased resistance to thermal inactivation. Even the relatively mild screening conditions applied were able to reduce the activity of wild-type P ⁄ CAF by approxi- mately 90%, allowing the identification of mutations that detectably improved the thermostability. Of the 24 selected variants, twelve carried a single mutation and twelve carried two to four mutations. Twenty-four different amino acid mutations were identified in total (Table 1). The observation that the screening of a rela- tively small number of library members (1000) lead to a high percentage of variants displaying higher thermo- stability may be a reflection of P ⁄ CAF being mono- meric under the assay conditions, whereas it is likely to be part of multi-subunit complexes in vivo. Most mutations were charge neutral. Three mutations intro- duced a charge, four mutations removed a charge and two mutations switched a positive and negative charge. At this stage, one of the mutants (V582A) was intro- duced into the pREST-P ⁄ CAF vector, purified and shown to have an apparent melting temperature that was 3 °C higher than the wild-type catalytic domain of P ⁄ CAF (Fig. 3), demonstrating the feasibility of creat- ing thermostable P ⁄ CAF mutants by directed evolu- tion. Second round of directed evolution To further increase the thermostability of P ⁄ CAF, a second round of evolution was performed by randomly recombining all 24 selected P ⁄ CAF mutants using DNA shuffling. Here, a more stringent heat challenge was applied, with a 75 min incubation at 37 °C. Screening of 700 variants yielded seven mutants with enhanced resistance to thermal inactivation compared to the best mutant (V582A) selected in the first round of directed evolution. Sequencing revealed that four double and three triple mutants had been selected, that all seven variants were combinations of mutations selected in the first round of directed evolution (Table 1) and that no additional mutations were intro- duced during the shuffling procedure. Characterization of selected P/CAF variants To investigate the thermostability of the selected mutants in more detail, two double and one triple mutant enzymes were expressed and purified to homo- geneity. Differential scanning calorimetry (DSC) was used to measure directly the thermal denaturation of the proteins, giving the apparent melting temperature (T m ). All P ⁄ CAF enzymes tested showed irreversible thermal unfolding, prohibiting the calculation of the free energy (DG) of unfolding. All selected variants unfolded at higher temperatures than wild-type P⁄ CAF, with the L503P ⁄ D601G ⁄ Y612C and V582A ⁄ D639E mutants having the highest apparent denaturation temperatures (Fig. 3 and Table 2). At this stage, we aimed to deter- mine the contribution of the individual mutations to thermostability, and constructed the L503P, D601G and D639E mutants by site-directed mutagenesis. DSC of the single mutants showed that D601G, V582A and D639E made a large contribution to the increased denaturation temperature of the selected P ⁄ CAF variants (Fig. 3). The Y612C mutation repro- ducibly had the opposite effect, slightly lowering the apparent melting temperature. This mutant was still selected because it is stabilizing compared to the wild- type enzyme, under the screening conditions, where there is acetyl-CoA. By contrast, the DSC measure- ments are made in the absence of acetyl-CoA, explain- ing the effect of the Y612C mutation. Table 1. Mutations identified in P ⁄ CAF variants displaying higher thermostability under screening conditions. The second column gives the relative solvent accessibility score as calculated by the software ASA-VIEW [35]. Mutant a Solvent accessibility score (%) First round (22 °C) L503P ⁄ D601G 50-91 N504I ⁄ Q519L ⁄ V572A ⁄ V582A 100-14-8-94 I511T ⁄ M529I 7-16 Q519R 14 H524Q 79 T535A ⁄ D551G 38-63 K542E ⁄ D639E 60-56 V582A 94 V582D 94 H592R 42 H600R 54 F605L 0 E611K 65 Y612C 87 K627R ⁄ D639E 36-56 D639V 56 E649D 77 P655R – b Second round (37 °C) L503P ⁄ D601G ⁄ Y612C 50-91-87 K542E ⁄ D601G ⁄ Y612C 60-91-87 D551G ⁄ V582A ⁄ D639E 63-94-56 V582A ⁄ Y612C 94-87 V582A ⁄ D639E 94-56 V582D ⁄ D639V 94-56 Y612C ⁄ P655R 87- b a Some mutations were found more than once: L503P ⁄ D601G (·2), V582A (·4), Y612C (·2) and K627R ⁄ D639E (·2). b Pro655 is not vis- ible in the structure. H. Leemhuis et al. Stabilizing a human histone acetyltransferase FEBS Journal 275 (2008) 5635–5647 ª 2008 The Authors Journal compilation ª 2008 FEBS 5637 Thermal inactivation Resistance to thermal inactivation of the P ⁄ CAF mutants was determined by heating protein samples for 5 min at various temperatures and measuring resi- dual activity. In this case, the reduction in activity corresponds to the percentage of enzyme molecules undergoing irreversible inactivation. We found that the temperature at which enzymes lost 50% of their activ- ity (T 50 ) was increased for all mutants examined (Fig. 4 and Table 2), with the triple mutant L503P ⁄ D601G ⁄ Y612C having the highest T 50 value. Note that these T 50 temperatures are close to the denaturating temperatures for the corresponding enzymes, and that the stabilizing effect (as measured in °C) for both methods is similar (Table 2). Interestingly, the Y612C mutant displayed an increased T 50 tempera- ture, consistent with selection in the thermostability screen, despite its lower apparent T m temperature. Next, we investigated whether the cofactor acetyl- CoA stabilizes P ⁄ CAF against thermal inactivation, as observed for two other HATs [29,30], and evaluated whether the effect is comparable for wild-type and mutant enzymes. Inactivation experiments were repeated in the presence of saturating acetyl-CoA (100 lm), revealing a large stabilizing effect for acetyl- CoA (5.4–8.7 °C) for all enzymes examined (Fig. 4 and Table 2). Comparison of the T 50 and T 50 AcCoA values indicated that acetyl-CoA binding is particularly stabi- lizing for the V582A, Y612C and V582A ⁄ D639E mutants. The time taken for temperature-dependent inactiva- tion was assessed by measuring the activity half-life (t 1 ⁄ 2, 48 °C ), which was determined at 48 °C. All mutants showed longer activity half-lives than wild- type P ⁄ CAF (Table 2), with the most stable mutants having 60- to 70-fold larger t 1 ⁄ 2, 48 °C values, broadly following the trend seen for the T 50 and T m tempera- tures. The stabilizing effects of single mutations are A B Fig. 3. Thermal denaturation traces mea- sured by differential scanning calorimetry. (A) Multiple P ⁄ CAF mutants and (B) single P ⁄ CAF mutants (compared to wild-type, wt). Conditions: 20 l M P ⁄ CAF protein in 50 m M sodium phosphate (pH 7.5), 150 mM NaCl and a scan rate of 1 °CÆmin )1 . Table 2. Stability parameters of purified P ⁄ CAF and selected mutants. Measurements were performed under the following con- ditions: 50 m M sodium phosphate (pH 7) and 150 mM NaCl. T m , apparent melting temperature; T 50 , temperature at which half of the initial activity is lost in 5 min; T 50 AcCo , temperature at which half of the initial activity is lost in 5 min in the presence of acetyl-CoA; t 1 ⁄ 2, 48 °C , activity half-life at 48 °C. Errors for T m and T 50 values are less than 0.5 °C. T m b, c (°C) T 50 (°C) T 50 AcCoA (°C) t 1 ⁄ 2, 48 °C (min) Wild-type 46.2 48.5 53.9 5.0 ± 0.1 L503P 47.0 48.1 54.9 9.9 ± 0.4 V582A 49.3 52.4 59.9 60 ± 3 D601G 52.3 53.5 59.3 315 ± 10 Y612C 45.0 49.4 57.5 17 ± 1 D639E 49.1 50.3 56.3 32 ± 2 Y612C ⁄ P655R a 49.5 50.5 57.2 35 ± 5 L503P ⁄ D601G ⁄ Y612C a 54.2 55.4 61.4 315 ± 12 V582A ⁄ D639E a 52.4 53.2 61.9 347 ± 16 a Mutants selected in second round of directed evolution. b For thermal inactivation curves, see Fig. 4. c Measured by differential scanning calorimetry. Stabilizing a human histone acetyltransferase H. Leemhuis et al. 5638 FEBS Journal 275 (2008) 5635–5647 ª 2008 The Authors Journal compilation ª 2008 FEBS approximately additive in the double and triple mutants for all stability measurements, as expected for mutations that are far apart in the structure. Overall, the inactivation experiments clearly demonstrate that the selected P ⁄ CAF mutants have a strongly enhanced resistance towards thermal inactivation. Catalytic properties and specificity of the P/CAF enzymes The kinetic parameters of the wild-type and mutant P ⁄ CAF enzymes for acetylation of the histone H3 and H4 peptides were determined using a continuous assay and synthetic peptide substrates. Wild-type P ⁄ CAF has k cat and K M values of 12 min )1 and 53 lm with the H3 peptide and 0.29 min )1 and 194 lm with the H4 pep- tide, whereas the K M for acetyl-CoA was 0.28 lm (Table 3). Note that the H3 peptide is a much better substrate for the enzyme, with k cat ⁄ K M of 3774 s )1 Æm )1 versus 25 s )1 Æm )1 for the H4 peptide, in agreement with histone H3 being the physiological substrate for P ⁄ CAF [31]. The mutations had no significant effect on the k cat H3 values but the V582A and Y612C muta- tions increased the K M H3 value by ten-fold (Table 3). An overlay of the P ⁄ CAF structure and the Tetrahy- mena HAT, with a bound H3 peptide shows that Tyr612 is located in the binding groove for the peptide substrate, which may explain the higher K M H3 observed with the Y612C mutant. By contrast, the basis of the high K M H3 of the V582A mutant is unclear because Val582 is far away from the peptide binding groove. The Y612C mutation reduced the k cat H4 by three-fold, whereas the other mutations had no signifi- cant effect on k cat values (Table 3). Histone acetylation at distinct histone residues is thought to have variable functional effects. We there- fore examined whether the thermostable P ⁄ CAF mutants affected the specificity of HAT activity. Recombinant histone H4 [32,33] (containing minimal endogenous post-translational modifications) was incu- bated with wild-type and four single mutant P ⁄ CAF enzymes and the specificity of acetylation was assessed by western blotting using antibodies specific for A B D C Fig. 4. Thermal inactivation curves of P ⁄ CAF and selected mutants. In the absence (A, B) and presence (C, D) of 100 l M ace- tyl-CoA, used to determine T 50 (Table 2). Conditions: 10 lM P ⁄ CAF protein in 50 m M sodium phosphate (pH 7.5) and 150 mM NaCl was incubated for 5 min at various temperatures before measuring the residual activity by ELISA. The activity measured without a heat challenge was set to 100%. The values derived from these curves are given in Table 2. H. Leemhuis et al. Stabilizing a human histone acetyltransferase FEBS Journal 275 (2008) 5635–5647 ª 2008 The Authors Journal compilation ª 2008 FEBS 5639 acetylation at distinct histone H4 lysines (K5, K8, K12 and K16). Figure 5 shows the specificity profiles of wild-type P ⁄ CAF and its mutants, normalized using the H4K16ac (i.e. the most physiologically abundant acetyl isoform). Broadly, the wild-type and mutant enzymes yield very similar patterns of lysine specificity, indicating that the individual mutations do not impact on substrate recognition. This may be expected because the immediate sequence environments of many of these residues are similar [e.g. for H4K5, 8 and 12 GK(ac)-G]. Discussion Location of mutations in the P/CAF structure Analysis of the location of the thermostabilizing mutations identified, and comparison with homolo- gous HAT sequences, shows that they are scattered throughout the enzyme structure (Fig. 6), largely occurring at nonconserved amino acid residues, with only two conserved residues being mutated (see Fig. S1). Close examination of the P ⁄ CAF crystal structure [34] reveals that almost all of these stabiliz- ing mutations are on the surface of the enzyme. This observation is reinforced by comparison of the distri- butions of solvent accessible residues in the entire proteins versus the residues selected by directed evolu- tion. The proportion of exposed residues (calculated with the programme asa-view) [35] is markedly increased in the selected set (see Fig. S2 and Table S1). Together with the analysis of the individual mutations, this suggests that the raised thermostablity is brought about by optimization of surface residues. Table 3. Kinetic parameters of the wild-type and mutant P ⁄ CAF enzymes at 25 °C and pH 7.5 in 150 mM Mes buffer. k cat H3 (min )1 ) K M H3 (lM) k cat H4 (min )1 ) K M H4 (lM) K M AcCoA (lM) a Wild-type 12 ± 1 53 ± 10 0.29 ± 0.02 190 ± 30 0.28 ± 0.03 L503P 12 ± 1 54 ± 14 0.32 ± 0.02 210 ± 30 0.33 ± 0.03 V582A 11 ± 1 560 ± 80 0.29 ± 0.02 500 ± 50 0.36 ± 0.06 D601G 12 ± 1 55 ± 11 0.33 ± 0.02 250 ± 20 0.34 ± 0.02 Y612C 11 ± 1 165 ± 29 0.11 ± 0.01 120 ± 30 0.41 ± 0.03 D639E 12 ± 1 39 ± 10 0.35 ± 0.03 150 ± 40 0.27 ± 0.02 Y612C ⁄ P655R 12 ± 1 62 ± 15 0.21 ± 0.01 140 ± 20 0.29 ± 0.03 L503P ⁄ D601G ⁄ Y612C 12 ± 1 64 ± 15 0.18 ± 0.02 110 ± 30 0.27 ± 0.03 V582A ⁄ D639E 13 ± 1 460 ± 60 0.37 ± 0.01 310 ± 20 0.34 ± 0.06 a K M AcCoA was determined using the H3 peptide. Fig. 5. Lysine specificity of wild-type (wt) and mutant P ⁄ CAF enzymes on histone H4. Acetylation of lysines 4, 8, 12 and 16 of histone H4 was measured by western blotting using site specific anti-acetyl sera. The degree of acetylation was normalized to that detected at lysine 16, which was given a value of 1.0. Fig. 6. Location of thermostabilizing mutations. Cartoon represen- tation of the catalytic domain of P ⁄ CAF (crystal structure 1CM0 of the Protein Data Bank) [34]. Residues mutated in variants with higher thermostability are indicated by sticks. Magenta indicates the residues shown to be stabilizing as a single mutant and grey indicates that the residue was found mutated only in combination with one or more other mutations. The catalytic glutamate residue is shown in green and the bound coenzyme A molecule is shown in black. Stabilizing a human histone acetyltransferase H. Leemhuis et al. 5640 FEBS Journal 275 (2008) 5635–5647 ª 2008 The Authors Journal compilation ª 2008 FEBS The high rate of surface mutations is consistent with the likely scenario that P⁄ CAF is part of larger multi- subunit complexes in vivo. Directed evolution of the monomeric P ⁄ CAF under the conditions described here then replaces the residues originally responsible for potential binding interactions to partner proteins by stabilizing residues. The mutations that are most effective in raising the thermostability of the catalytic domain of P ⁄ CAF (V582A, D601G and D639E) are discussed in more detail below. Mutation V582A introduced a smaller alanine residue at the position of Val582, a residue strictly conserved among HATs. This residue is in the vicinity of the coenzyme A molecule bound to the enzyme and is solvent exposed, indicating that the V582A mutation increases thermostability by lowering the hydrophobicity of the protein’s surface. The analo- gous V582D mutation is also likely to derive its stabilizing effect as a result of reduced surface hydro- phobicity. Alternatively, the effect of this mutation may be explained by modulating the surface charge distribution, electrostatic interactions, or a combina- tion of these effects. Similarly, the thermostabilizing mutation D601G is located in a short loop connecting an a-helix and a b-strand at the surface of the protein, with the Asp601 side-chain solvent being exposed. The observation that the D601G mutation is stabilizing is noteworthy because the introduction of glycines is usu- ally not considered to be stabilizing [20]. Asp601 is not conserved among GCN5 HAT enzymes, and a glycine at the equivalent position is found in a few other HATs (see Fig. S1). The source of stabilization is likely to arise from optimization of the surface charge distribution by removing the negatively charged aspar- tate side-chain. Alternatively, the release of conforma- tional strain allowed by the extra flexibility of the glycine residue may lead to better protein packing and increased thermostability. The latter mechanism of sta- bilization has been discussed in more detail by Veille and Zeikus [10]. A D639E mutation significantly increased the ther- mostability of P⁄ CAF. This aspartate residue is found in most GCN5 HATs (see Fig. S1) and is part of a loop region at the surface of the enzyme with its side chain solvent being exposed. Different conformations have been observed for this loop in crystal structures of GCN5 HAT enzymes and a recent study demon- strated that the movement of this loop correlates with the different stages of the acetylation reaction [36]. A possible explanation for the stabilizing effect of D639E is that the longer glutamate side chain provides more opportunities for salt bridge formation on the surface of the protein. Implications for protein thermostability engineering Generally, only a few mutations are sufficient to increase the thermostability of a protein by as much as tens of degrees Celsius [37–40] but the prediction of the these mutations proves to be a challenge. One might assume (e.g. for structure-based computational approaches that are especially advantageous when no straightforward screening or selection systems are available) that surface residues should not be targeted to increase thermal stability because their interactions with the solvent were expected to be similar in the native and unfolded state of the protein. Instead, inter- nal residues have been preferred targets as in the com- putational optimization of protein structures [41–43]. Our study suggests that surface residues play a more important role in P ⁄ CAF: such mutations occurred in residues with a clearly higher solvent accessibility rela- tive to the average residue of this protein: 51.7% ver- sus 34.1%; scale 0–100%; as calculated with asa-view [35]. For the single mutations shown to be stabilizing in the present study (eleven in total, excluding P655R, which showed no electron density in the crystal struc- ture), the solvent accessibility was even higher at 59.6% (see Fig. S2 and Table S1). Other directed evo- lution studies aimed at the generation of thermostable proteins also frequently identify surface mutations [27]; seven out of eight in the case of an esterase [44], three out of three for an a-glucan phosphorylase [24] and ten out of 12 for a phosphite dehydrogenase [45]. Computational optimization of surface charge–charge interactions was able to predict site-directed mutants with increased thermostabilities for five small proteins (with < 100 amino acid residues) [46]. This preference for surface mutations may be explained by (a) the rela- tively high percentage of residues located at the protein surface, (b) surface residues forming proportionally fewer interactions than ‘internal’ residues, and there- fore being less likely to cause detrimental effects that offset gains by mutation, or (c) that the surface is rela- tively important to thermostability of proteins. In addition, (d) protein surfaces have been suggested to be important for thermostability of proteins showing irreversible unfolding [47] as in the case with P ⁄ CAF. This scenario may involve partial unfolding of the protein surface structure that would be addressed by surface mutations. Sequence comparison approaches in the design of proteins with enhanced stability are based on the assumption that there is a positive correlation between the conservation of a residue and its contribution to the stability of the protein. Thus, substituting poorly H. Leemhuis et al. Stabilizing a human histone acetyltransferase FEBS Journal 275 (2008) 5635–5647 ª 2008 The Authors Journal compilation ª 2008 FEBS 5641 conserved residues for more conserved residues is expected to increase the thermostability of a protein, as shown for GroEL minichaperone [14], immuno- globulin domains [48], phytase [49] and a few other proteins [20,50,51]. To investigate whether the stabi- lized P ⁄ CAF mutants carry mutations predicted by sequence comparison, the sequences of 15 homologous HAT proteins, sharing approximately 25% sequence identity, were aligned (see Fig. S3). We then matched the stabilizing single mutants with the sequence align- ment. The sequence comparison showed that 31 resi- dues out of 167 (or 19%) in P ⁄ CAF, were different from the amino acid most frequently found at this position. Thus, according to the sequence comparison approach, these 31 residues are the most likely to be mutated. However, of the twelve stabilizing mutations found by directed evolution, only the D601G muta- tion was marginally predominant (with five glycine versus three aspartate residues at this position). All other stabilizing mutations introduced less conserved residues (H600R, F605L, D639E and P655R) or resi- dues not seen in any of the HATs (Q519R, H524Q, V582A, H592R, E611K, Y621C and E649D). Muta- tion V582A even substituted the completely conserved Val582. These results emhasize the effectiveness of directed evolution in the identification of thermostabi- lizing mutants that cannot be predicted by rational protein design. Because the effects of stabilizing muta- tions are often additive, a combination of sequence comparison and directed evolution may create even more stable proteins by combining the advantages of both methods. Conclusions Proteins have evolved to be stable and biologically active under conditions imposed by the cellular envi- ronment and the habitat of the organism; however, there is little evolutionary advantage in being more stable than required. Because the majority of muta- tions are destabilizing, most proteins will be only mar- ginally stable [52–54]. This implies that there is ample sequence flexibility available to stabilize proteins, whilst retaining their catalytic capacity. In the present study, we have shown that mutations stabilizing the catalytic domain of the human histone acetyltransfer- ase P ⁄ CAF are readily identified by directed evolu- tion. These mutations have either no or minimal effects on the catalytic properties and specificity of this enzyme. The directed evolution method described is able to generate stable histone modifying enzymes in a straightforward procedure. Furthermore, histone modifying enzymes are potential targets for drug screening in ‘epigenetic therapies’ for a range of dis- eases, notably cancers and acute myeloid leukaemia [55–58]. A more thermostable target enzyme with identical catalytic properties will facilitate high- throughput drug screening and allow biochemical experiments to be carried out in the absence of poten- tial binding partners that are stabilizing the protein in vivo. The ELISA screening approach described in the present study is easily adapted to investigate other histone modifying enzymes simply by using another appropriate histone substrate and antibody, which are widely available from commercial suppliers. This there- fore represents a general protocol to improve the sta- bility of a wide range of histone modification enzymes for in vitro applications. Experimental procedures Antibodies and peptides Polyclonal rabbit antibodies specific for the recognition of acetylated lysine residues in histone tails (H4K5ac, H4K8ac, H4K12ac and H4K16ac) were kindly provided by Bryan Turner (University of Birmingham, UK). Goat anti- (rabbit IgG) serum conjugated to horseradish peroxidase was purchased from Sigma (St Louis, MO, USA). Histone tail peptides were synthesized by the PNAC facility (Cam- bridge University, UK): H3, ARTKQTARKSTGGKAPR KQLC, and H4, SGRGKGGKGLGKGGAKRHRKV GGK. The H4 peptide is biotinylated at its C-terminus to allow attachment to streptavidin coated microtitre plates. Expression and purification All (mutant) P ⁄ CAF proteins (20 kDa) were expressed in Escherichia coli BL21(DE3) from pRSET-P ⁄ CAF in liquid medium (10 gÆL )1 NaCl, 10 gÆL )1 yeast extract and 20 gÆL )1 bacto tryptone) with 100 mgÆL )1 ampicillin at 25 °C. This vector expresses the catalytic domain of P ⁄ CAF. The proteins were purified by cation exchange and size exclusion chromatography, as described previously [59]. In the latter column, P ⁄ CAF eluted as a monomer (at con- centrations above those used in the stability and catalytic assays), suggesting that any effects of mutations on the oligomerization state can safely be excluded, thus simplify- ing the analysis of stability-conferring mutations. DNA manipulation The gene encoding the catalytic domain (amino acids 492– 658) of P ⁄ CAF [60] was amplified from pRSET-P ⁄ CAF using the primers P ⁄ CAF-for-BamHI and P ⁄ CAF-rev-PstI. The resulting PCR product was restricted with BamHI and Stabilizing a human histone acetyltransferase H. Leemhuis et al. 5642 FEBS Journal 275 (2008) 5635–5647 ª 2008 The Authors Journal compilation ª 2008 FEBS PstI and cloned into pMalC2x (New England Biolabs, Beverly, MA, USA), yielding plasmid pMal-P ⁄ CAF. This construct expresses P ⁄ CAF as C-terminal fusion to the maltose binding protein (see Fig. S4). DNA sequencing was carried out with the T7 or Seq oligonucleotides. L503P, D601G and D639E mutations were introduced in pRSET- P ⁄ CAF using the QuickChange system (Stratagene, La Jolla, CA, USA). Oligonucleotide sequences are provided in the Table S2. First generation library construction In the first round of directed evolution, the catalytic domain of P ⁄ CAF was expressed as C-terminal fusion to maltose binding protein to minimize the effects of the random mutations on the amount of protein expressed. Random mutations were introduced in the gene encoding P ⁄ CAF by error-prone PCR amplification in the presence of manganese chloride. Additional details on directed evo- lution procedures are provided elsewhere [61]. Optimal error-prone PCR conditions were determined by sequencing P ⁄ CAF genes amplified at various MnCl 2 concentrations. As expected, the number of nucleotide mutations increased with increasing MnCl 2 concentrations with a clear prefer- ence for T > C and A > G substitutions. The library con- structed with 0.075 mm MnCl 2 was used for screening because it had one to two nucleotide mutations per gene, and two to four changes per 1000 bp. The gene for P ⁄ CAF was amplified from plasmid pMal-P ⁄ CAF using primers pMal-forII and pMal-revII (see Table S2), which anneal at the BamHI and PstI restriction sites used for cloning; for a schematic view of the gene and its flanking regions, see the Fig. S4. PCRs (50 lL) contained: 1· Taq DNA polymerase buffer, 0–0.5 mm MnCl 2 , 0.4 mm of each dNTP, 10 ng of pMal-P ⁄ CAF template, primers at 0.5 lm and 4 U Taq DNA Polymerase (New England Biolabs). The PCR pro- gram used was: 1 min at 94 °C; 30 cycles each comprising 45 s at 94 °C, 45 s at 54 °C and 45 s at 72 ° C; followed by 5 min at 72 °C. PCR products were digested with BamHI and PstI, purified with a QIAquick PCR purification Kit (Qiagen, Valencia, CA, USA) and cloned into pMalC2x by incubating overnight with T4 DNA ligase (New England Biolabs) at 16 °C. Ligated plasmids were transformed into E. coli DH5a cells (Invitrogen, Carlsbad, CA, USA) and plated onto LB agar with 100 mgÆL )1 ampicillin. Plasmids were purified from pooled transformants and stored at )20 °C. Second generation library construction Plasmid DNA of all 24 P ⁄ CAF variants selected in the first round of directed evolution were mixed and used as tem- plates in a PCR with Pfu-DNA polymerase (Stratagene) with primers pMal-for and pMal-rev. These primers anneal approximately 100 bp upstream and downstream of the gene for P ⁄ CAF (see Fig. S4). The PCR product, purified with a QIAquick PCR Purification Kit (Qiagen) was digested with DNAse (3 U; Promega, Madison, WI, USA) at 15 °C for 3, 4 and 5 min. Digestion was stopped by add- ing EDTA to 10 mm. Following separation on an agarose gel (2%), fragments between 50 and 100 bp were isolated with the Qiagen II gel extraction kit (Qiagen). A re-assem- bly reaction (50 lL) contained 100 ng of DNA fragments, 0.4 mm of each dNTP, 1· Pfu reaction buffer and 2.5 U Pfu DNA polymerase. The re-assembly reaction (1 min at 94 °C; 70 cycles each comprising 40 s at 94 °C, 30 s at 50 °C, 30 s at 94 °C; followed by 4 min at 72 °C) yielded a product of expected size ( 750 bp) plus a smear of smaller and larger products. Aliquots of this re-assembly reaction were used as PCR templates to amplify the shuffled P ⁄ CAF genes using Pfu-DNA polymerase and primers pMal-forII and pMal-revII, obtaining a product of the expected size ( 550 bp). This fragment was digested with BamHI and PstI and cloned into pRSET (Invitrogen). Ligated plasmids were transformed into E. coli DH5a cells, plated onto LB agar with 100 mgÆL )1 ampicillin, and plasmids were purified from the pooled transformants to yield the second genera- tion library. Screening procedure The plasmid libraries were transformed into E. coli BL21(DE3), plated on LB agar supplemented with 100 mgÆL )1 ampicillin and resulting colonies were trans- ferred to 200 lL of LB medium in 96-well plates. A sche- matic view of the screening procedure is provided in Fig. 2. After overnight incubation at 37 °C and shaking at 750 r.p.m., 25 lL was transformed to a fresh 96-well plate containing 200 lL of LB and 0.25 mm isopropyl thio-b-d-galactoside followed by incubation at 37 °C (750 r.p.m. shaking speed) for 4 h. Cells were harvested by centrifugation (4000 g). To release the P ⁄ CAF activity, cells were broken by resuspension in a mixture of BugBust- er (Novagen, Madison, WI, USA) and demineralized water (40 : 60, v ⁄ v; total volume of 20 lL) and incubated at room temperature (20–25 °C) for 15 min. The plates were subsequently incubated for 2 h at 4 and 22 ° C, respec- tively, or for 75 min at 37 °C. The remaining HAT activity after exposure to elevated temperatures was determined by ELISA, as described below. Plasmid DNA of clones expressing a more stable P ⁄ CAF was isolated and used to re-transform E. coli DH5a cells to verify the apparent increase in thermal stability. ELISA procedure ELISA plates were prepared by incubating clear 96-well streptavidin coated microtitre plates (SigmaScreen; Sigma) with biotinylated H4 peptide (0.5 lgin50lL NaCl ⁄ P i per well) at room temperature for 30 min. After rinsing with H. Leemhuis et al. Stabilizing a human histone acetyltransferase FEBS Journal 275 (2008) 5635–5647 ª 2008 The Authors Journal compilation ª 2008 FEBS 5643 water (·4), plates were blocked with BSA in NaCl ⁄ P i (3%, w ⁄ w) at room temperature for 1 h. Plates were then rinsed with water (·4) and HAT reactions were started by the addition of reaction buffer [5 · reaction buffer: 250 mm Tris ⁄ HCl (pH 8.0), 5 mm dithithreitol, 0.5 m m EDTA and 50% glycerol (v ⁄ v), 200 lm acetyl-CoA (Sigma)] and either purified enzyme or lysate of E. coli cells expressing P ⁄ CAF protein. Reactions were continued for 10–45 min at room temperature and stopped by washing (·5) with water. The degree of acetylation was determined using an antibody specific for acetylated K8 in the H4 peptide, by diluting the antibodies 200-fold in NaCl ⁄ P i with 3% BSA and incubat- ing at room temperature for 1 h. Plates were washed with NaCl ⁄ P i ⁄ 0.5 m NaCl ⁄ 1% Tween-20 (·2), NaCl ⁄ P i ⁄ 1% Tween-20 (·2) and water (·3). The secondary antibody, an anti-(rabbit IgG) horseradish peroxidase conjugate, was added at a dilution of 1000-fold in NaCl ⁄ P i with BSA (3%, w ⁄ w) and incubated for 30 min. Following washing with NaCl ⁄ P i ⁄ 1% Tween-20 (·3) and water (·4), 100 lLof peroxidase substrate (tetramethylbenzidine; KPL, Silver Spring, MD, USA) was added and incubated for 5–10 min. This peroxidase reaction was stopped by the addition of sulfuric acid (50 lL, 1 m) and A 450 was measured. Under the conditions described above, the increase in absorbance obtained from the ELISA is linear (for the wild-type with- out the exposure to the high temperature) for the time between 0 and 30 min for acetylation of K8 in the H4 peptide. Continuous HAT assay Progress curves as a measure of HAT activity of wild-type and mutant P ⁄ CAFs were followed using a coupled enzyme assay [62]. HAT activity generates CoA as a byproduct that is converted back to acetyl-CoA by pyruvate dehydroge- nase, which is accompanied by NAD + reduction to NADH, thus increasing A 340 . An example of a progress curve is provided in the Fig. S3. Reactions were carried out in 96-well plates at 25 °C. Reaction mixtures (300 lL) contained Mes buffer (pH 7.5, 100 mm), H3 peptide (0– 500 lm) or H4 peptide (0–700 lm), acetyl-CoA (0– 1000 lm), NAD + (0.2 mm), thiamine pyrophosphate (0.2 mm), MgCl 2 (5 mm), dithiothreitol (1 mm), pyruvate (2.4 mm) and pyruvate dehydrogenase (0.3 UÆmL )1 ; Sigma). Reactions were initiated by the addition of P ⁄ CAF enzyme (i.e. the catalytic domain as expressed from pREST- P ⁄ CAF) at a concentration of 0.8–7 lm. Linear curves of reaction progress over time were measured at 340 nm, con- verted using a value of 5296 AbsÆM )1 for e*d. The value of e*d for NADH was determined from a calibration curve with pure NADH in our microtitre plate setup. The time traces gave initial rates that were fitted to the Michaelis– Menten equation. The measured rates were corrected for the spontaneous acetyl-CoA hydrolysis (obtained in the absence of enzyme). Stability assays Resistance to thermal inactivation was determined by incu- bating the P⁄ CAF protein in sodium phosphate (pH 7.5, 50 mm), NaCl (150 mm), dithithreitol (1 mm) and EDTA (1 mm) buffer. All stability assays were performed with P ⁄ CAF protein expressed from pRSET-P ⁄ CAF. Protein samples (25 lL, 10 l m P ⁄ CAF) were incubated at various temperatures in the range 0–67 °C for 5 min in a PCR machine followed by incubation on ice for at least 30 min. Residual activity was measured by ELISA. The activity measured without a heat challenge was set to 100%. We define the T 50 temperature as the temperature at which half of the initial activity is lost in 5 min. The activity half-life t 1 ⁄ 2, 48 °C of the proteins was determined at 48 °C by incu- bating the proteins for 0–1800 min at 48 °C, followed by incubation on ice. Residual activity was determined by ELISA and the activity half-life (t 1 ⁄ 2, 48 °C ) is defined as the time in which the activity is reduced by 50%. DSC Thermal unfolding was measured using the MicroCal VP-DSC microcalorimeter (MicroCal Inc., Northampton, MA, USA), with a cell volume of 0.5 mL. Experiments were carried out at a scan rate of 1 °CÆmin )1 from 2–75 °C at a constant over-pressure of 1.79 bar (26 psi). Samples were degassed prior to the scan and contained 20 lm (0.40 mgÆmL )1 )P⁄ CAF protein (expressed from pRSET- P ⁄ CAF) in sodium phosphate (pH 7.5, 50 mm), NaCl (150 mm), dithiothreitol (1 mm) and EDTA (1 mm) buffer. Samples were dialyzed against this buffer and the reference cell contained the dialysis buffer of the last dialysis step. The apparent melting temperature (T m ) is defined as mid- point of thermal unfolding as seen in a DSC thermogram. Assessment of wild-type and mutant P/CAF substrate specificity Substrate specificity of wild-type and single substitution P ⁄ CAF mutants (expressed from pRSET-P ⁄ CAF) was assessed by western blotting using antibodies specific for acetylation at distinct histone H4 lysines (H4K5ac, H4K8ac, H4K12ac and H4K16ac). Histone acetyltransfer- ase assays were performed using 20 lgofE. coli expressed histone H4 substrate [32,33] and enzyme in reaction buffer [50 mm Tris ⁄ HCl (pH 8.0), 1 mm dithiothreitol, 0.1 mm EDTA, 200 lm acetyl-CoA] at 30 °C for 3 h. The reaction was halted by addition of SDS loading buffer, and sepa- rated on 15% acrylamide : bis-acrylamide (30 : 1) gel, prior to transfer to nitrocellulose (Hybond-C; Amersham Phar- macia, Piscataway, NJ, USA), western blotting and detec- tion using a fluorescently tagged anti-rabbit secondary (Licor, 800 nm). Stabilizing a human histone acetyltransferase H. Leemhuis et al. 5644 FEBS Journal 275 (2008) 5635–5647 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... Sequence alignment of the catalytic domain of 14 histone acetyltransferase sequences Stabilizing a human histone acetyltransferase Fig S2 Relative solvent accessibility of amino acid residues of the catalytic domain of P ⁄ CAF Fig S3 Example for a reaction progress curve as a measure of HAT activity Fig S4 The gene for P ⁄ CAF and its flanking regions Table S1 Relative solvent accessibility of amino acids of. .. 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CAF and its flanking regions. Table S1. Relative solvent accessibility of amino acids of the catalytic domain of P ⁄ CAF as calculated with the program asa-view. Table