Directed evolution of a histone acetyltransferase

Directed evolution of a histone acetyltransferase – enhancing thermostability, whilst maintaining catalytic activity and substrate specificity Hans Leemhuis1, Karl P. Nightingale1,2 and Florian Hollfelder1 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 (Tm). 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]. Mutanta 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 (T50) was increased for all mutants examined (Fig. 4 and Table 2), with the triple mutant L503P ⁄D601G ⁄Y612C having the highest T50 value. Note that these T50 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 T50 tempera- ture, consistent with selection in the thermostability screen, despite its lower apparent Tm 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 T50 and T50 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 (t1 ⁄ 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 t1 ⁄ 2, 48 �C values, broadly following the trend seen for the T50 and Tm 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 lM P ⁄ CAF protein in 50 mM 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 mM sodium phosphate (pH 7) and 150 mM NaCl. Tm, apparent melting temperature; T50, temperature at which half of the initial activity is lost in 5 min; T50 AcCo, temperature at which half of the initial activity is lost in 5 min in the presence of acetyl-CoA; t1 ⁄ 2, 48 �C, activity half-life at 48 �C. Errors for Tm and T50 values are less than 0.5 �C. Tm b, c (�C) T50 (�C) T50 AcCoA (�C) t1 ⁄ 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 ⁄ P655Ra 49.5 50.5 57.2 35 ± 5 L503P ⁄ D601G ⁄ Y612Ca 54.2 55.4 61.4 315 ± 12 V582A ⁄ D639Ea 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 kcat and KM 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 KM for acetyl-CoA was 0.28 lm (Table 3). Note that the H3 peptide is a much better substrate for the enzyme, with kcat ⁄KM of 3774 s)1Æm)1 versus 25 s)1Æm)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 kcat H3 values but the V582A and Y612C muta- tions increased the KM 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 KM H3 observed with the Y612C mutant. By contrast, the basis of the high KM H3 of the V582A mutant is unclear because Val582 is far away from the peptide binding groove. The Y612C mutation reduced the kcat H4 by three-fold, whereas the other mutations had no signifi- cant effect on kcat 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 lM ace- tyl-CoA, used to determine T50 (Table 2). Conditions: 10 lM P ⁄ CAF protein in 50 mM 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 o