MYST protein acetyltransferase activity requires active site lysine autoacetylation
2011; Springer Nature; Volume: 31; Issue: 1 Linguagem: Inglês
10.1038/emboj.2011.382
ISSN1460-2075
AutoresHua Yuan, Dorine Rossetto, Hestia Mellert, Weiwei Dang, Madhusudan Srinivasan, Jamel Johnson, Santosh Hodawadekar, Emily Chen Ding, Kaye D. Speicher, Nebiyu Abshiru, Rocco Perry, Jiang Wu, Chao Yang, Y. George Zheng, David W. Speicher, Pierre Thibault, Alain Verreault, F. Bradley Johnson, Shelley L. Berger, Rolf Sternglanz, Steven B. McMahon, Jacques Côté, Ronen Marmorstein,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle21 October 2011free access MYST protein acetyltransferase activity requires active site lysine autoacetylation Hua Yuan Hua Yuan Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Dorine Rossetto Dorine Rossetto Laval University Cancer Research Center, Hôtel-Dieu de Québec (CHUQ), Quebec City, Quebec, Canada Search for more papers by this author Hestia Mellert Hestia Mellert Department of Cancer Biology, The Kimmel Cancer Center, Thomas Jefferson Medical College, Philadelphia, PA, USA Search for more papers by this author Weiwei Dang Weiwei Dang Department of Cellular and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Madhusudan Srinivasan Madhusudan Srinivasan Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Jamel Johnson Jamel Johnson Department of Pathology and Laboratory Medicine, University of Pennsylvania, PA, USA Search for more papers by this author Santosh Hodawadekar Santosh Hodawadekar Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Emily C Ding Emily C Ding Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Kaye Speicher Kaye Speicher Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Nebiyu Abshiru Nebiyu Abshiru Département de Chimie, Université, de Montréal, Montreal, Quebec, Canada Search for more papers by this author Rocco Perry Rocco Perry Department of Cellular and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Jiang Wu Jiang Wu Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author Chao Yang Chao Yang Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author Y George Zheng Y George Zheng Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author David W Speicher David W Speicher Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Pierre Thibault Pierre Thibault Département de Chimie, Université, de Montréal, Montreal, Quebec, Canada Search for more papers by this author Alain Verreault Alain Verreault Département de Pathologie et Biologie Cellulaire, Institute for Research in Immunology and Cancer, Université de Montréal, Succursale Centre-Ville, Montreal, Quebec, Canada Search for more papers by this author F Bradley Johnson F Bradley Johnson Department of Pathology and Laboratory Medicine, University of Pennsylvania, PA, USA Search for more papers by this author Shelley L Berger Shelley L Berger Department of Cellular and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Rolf Sternglanz Rolf Sternglanz Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Steven B McMahon Steven B McMahon Department of Cancer Biology, The Kimmel Cancer Center, Thomas Jefferson Medical College, Philadelphia, PA, USA Search for more papers by this author Jacques Côté Jacques Côté Laval University Cancer Research Center, Hôtel-Dieu de Québec (CHUQ), Quebec City, Quebec, Canada Search for more papers by this author Ronen Marmorstein Corresponding Author Ronen Marmorstein Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Hua Yuan Hua Yuan Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Dorine Rossetto Dorine Rossetto Laval University Cancer Research Center, Hôtel-Dieu de Québec (CHUQ), Quebec City, Quebec, Canada Search for more papers by this author Hestia Mellert Hestia Mellert Department of Cancer Biology, The Kimmel Cancer Center, Thomas Jefferson Medical College, Philadelphia, PA, USA Search for more papers by this author Weiwei Dang Weiwei Dang Department of Cellular and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Madhusudan Srinivasan Madhusudan Srinivasan Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Jamel Johnson Jamel Johnson Department of Pathology and Laboratory Medicine, University of Pennsylvania, PA, USA Search for more papers by this author Santosh Hodawadekar Santosh Hodawadekar Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Emily C Ding Emily C Ding Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Kaye Speicher Kaye Speicher Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Nebiyu Abshiru Nebiyu Abshiru Département de Chimie, Université, de Montréal, Montreal, Quebec, Canada Search for more papers by this author Rocco Perry Rocco Perry Department of Cellular and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Jiang Wu Jiang Wu Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author Chao Yang Chao Yang Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author Y George Zheng Y George Zheng Department of Chemistry, Georgia State University, Atlanta, GA, USA Search for more papers by this author David W Speicher David W Speicher Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Pierre Thibault Pierre Thibault Département de Chimie, Université, de Montréal, Montreal, Quebec, Canada Search for more papers by this author Alain Verreault Alain Verreault Département de Pathologie et Biologie Cellulaire, Institute for Research in Immunology and Cancer, Université de Montréal, Succursale Centre-Ville, Montreal, Quebec, Canada Search for more papers by this author F Bradley Johnson F Bradley Johnson Department of Pathology and Laboratory Medicine, University of Pennsylvania, PA, USA Search for more papers by this author Shelley L Berger Shelley L Berger Department of Cellular and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Rolf Sternglanz Rolf Sternglanz Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Steven B McMahon Steven B McMahon Department of Cancer Biology, The Kimmel Cancer Center, Thomas Jefferson Medical College, Philadelphia, PA, USA Search for more papers by this author Jacques Côté Jacques Côté Laval University Cancer Research Center, Hôtel-Dieu de Québec (CHUQ), Quebec City, Quebec, Canada Search for more papers by this author Ronen Marmorstein Corresponding Author Ronen Marmorstein Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Author Information Hua Yuan1, Dorine Rossetto2,‡, Hestia Mellert3,‡, Weiwei Dang4,‡, Madhusudan Srinivasan5, Jamel Johnson6, Santosh Hodawadekar1, Emily C Ding1,7, Kaye Speicher1, Nebiyu Abshiru8, Rocco Perry4, Jiang Wu9, Chao Yang9, Y George Zheng9, David W Speicher1, Pierre Thibault8, Alain Verreault10, F Bradley Johnson6, Shelley L Berger4, Rolf Sternglanz5, Steven B McMahon3, Jacques Côté2 and Ronen Marmorstein 1,7 1Gene Expression and Regulation Program, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA 2Laval University Cancer Research Center, Hôtel-Dieu de Québec (CHUQ), Quebec City, Quebec, Canada 3Department of Cancer Biology, The Kimmel Cancer Center, Thomas Jefferson Medical College, Philadelphia, PA, USA 4Department of Cellular and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA 5Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA 6Department of Pathology and Laboratory Medicine, University of Pennsylvania, PA, USA 7Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA 8Département de Chimie, Université, de Montréal, Montreal, Quebec, Canada 9Department of Chemistry, Georgia State University, Atlanta, GA, USA 10Département de Pathologie et Biologie Cellulaire, Institute for Research in Immunology and Cancer, Université de Montréal, Succursale Centre-Ville, Montreal, Quebec, Canada ‡These authors contributed equally to this work *Correspondence to: [email protected] The EMBO Journal (2012)31:58-70https://doi.org/10.1038/emboj.2011.382 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. Introduction Histone acetyltransferase (HAT) enzymes play important roles in the regulation of chromatin assembly, RNA transcription, DNA repair and other DNA-templated reactions through the lysine side-chain acetylation of histones and other transcription factors (Marmorstein, 2001; Wang et al, 2008). HATs fall into at least four different families based on sequence conservation within the HAT domain (Marmorstein and Trievel, 2009). This includes Gcn5/PCAF, p300/CBP, Rtt109 and MYST (named for the founding members MOZ, Ybf2/ Sas3, Sas2 and Tip60) families. The different HAT families contain a structurally conserved central region associated with acetyl-Coenzyme A (Ac-CoA) cofactor binding but distinct catalytic mechanisms and structurally divergent flanking regions that mediate different chromatin regulatory functions (Marmorstein and Trievel, 2009). Several recent proteomics studies also reveal that protein acetylation extends beyond histones to other nuclear proteins and even cytoplasmic proteins to regulate diverse biological processes including the regulation of the cell cycle, vesicular trafficking, cytoskeleton reorganization and metabolism (Choudhary et al, 2009; Smith and Workman, 2009; Spange et al, 2009; Wang et al, 2010; Zhao et al, 2010). In this way, protein acetylation may rival protein phosphorylation to mediate signal transduction pathways that control key cellular activities. The MYST proteins represent the largest family of HATs. They are conserved from yeast to man and mediate diverse biological functions including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development (Sapountzi and Cote, 2010). MYST proteins have also been shown to acetylate several non-histone substrates as highlighted by a recent protein acetylation microarray screen, revealing that the yeast Esa1 (yEsa1) containing NuA4 complex acetylates several cytoplasmic non-chromatin substrates known to be involved in the regulation of metabolism, the cell cycle and response to stress (Lin et al, 2009). MYST proteins have also been implicated in several forms of cancer and are particularly associated with chromosomal translocations in leukaemias (Avvakumov and Cote, 2007), also making them attractive drug targets. As with protein kinase enzymes (Morgan, 1995; Nolen et al, 2004), the activities and substrate selectivities of several MYST acetyltransferases are modulated by the binding of regulatory protein subunits. For example, yeast Sas2 requires binding to Sas4 and Sas5 for catalytic activity (Sutton et al, 2003), yEsa1 is found in the NuA4 complex and in a smaller Piccolo NuA4 complex to acetylate specific or global genomic locations, respectively (Allard et al, 1999; Boudreault et al, 2003), and human MOF (hMOF) is found in at least two distinct complexes, MSL and MOF-MSL1v1, that have indistinguishable activity on histone H4 K16, but differ dramatically in acetylating the non-histone substrate p53 (Li et al, 2009). Given the large number and diversity of MYST protein substrates and their involvement in various cellular processes, coupled with their modulation by other protein cofactors, we investigated the possibility that, also like protein kinases, the MYST proteins might be autoregulated by posttranslational modification. Results Structure of yEsa1 bound to a bisubstrate inhibitor reveals acetylation of a conserved lysine in the active site We previously reported on X-ray crystal structures of the HAT domain of the yEsa1 MYST protein bound to acetyl-CoA and CoA (Yan et al, 2000, 2002). In order to obtain insights into protein substrate binding by MYST proteins, we now report on the X-ray crystal structure of yEsa1 in complex with an H4K16CoA bisubstrate inhibitor (Wu et al, 2009) to 2.1 Å resolution (Figure 1A; Table I). The yEsa1 and CoA portion of the complex superimpose well with the previously reported yEsa1/CoA complex, although additional continuous electron density was observed emanating from the tip of CoA, which was used to model the linkage and H4K16 lysine side chain. Continuous electron density corresponding to backbone and side chain residues flanking H4K16 was not observed and we presume that these regions of the bisubstrate inhibitor are disordered in our structure. The H4K16 lysine side chain sits in a fairly open, largely hydrophobic, groove between the α2–β7 and β10–α4 loops flanking the Esa1 active site (Figure 1A). Figure 1.Structure of MYST protein active sites. (A) Superimposed structures of yEsa1 (pink) and hMOF (hMOF, green). The active sites are highlighted showing catalytic residues (glutamate and cysteine), the autoacetylated lysine and the H4K16CoA inhibitor bound to yEsa1. Numbering of residues corresponds to the yEsa1 sequence. (B) Structure of the yEsa1 active site. The simulated annealing omit map for K262Ac and the H4K16CoA inhibitor (1.5 sigma) is shown. Side chains that play catalytic roles and that make interactions with K262Ac are included as stick figures. The omit maps were generated by omitting a 10-Å radius around the selected atoms. (C) Sequence alignment of MYST protein active site residues and residues proximal to the autoacetylated lysine residues of yEsa1 and hMOF. Numbering above the sequences corresponds to the yEsa1 sequence. The conserved autoacetylated lysine residue is indicated with an inversed triangle and residues that interact with this lysine are highlighted with a star above the sequence. Only a selected set of MYST proteins are shown. (D) Structure of the hMOF active site. This figure is analogous to (B) except that corresponding hMOF residues are highlighted and the two alternate conformations of the α2–β7 loop harbouring the acetylated (K274Ac) and unacetylated (K274) residues, respectively, are highlighted with their corresponding simulated annealing omit maps (1.0 sigma). (E) Superposition of the yEsa1 and hMOF active sites. Numbering of residues corresponds to the hMOF sequence. (F) LC–MS/MS analysis of recombinant yEsa1 (upper panel) and hMOF (lower panel) tryptic peptides containing K262Ac and K274Ac, respectively. The extracted ion chromatogram (XIC) of the indicated peptide is shown on the left. A corresponding MS/MS spectrum of the 2+ charge state precursor is shown on the right with major b- and y-ions labelled. Download figure Download PowerPoint Table 1. Crystallographic data collection and refinement statistics yEsa1/H4K16CoA yEsa1/CoA yEsa1(E338Q)/CoA hMOF hMOF(E350Q) Data collection Space group I4132 I4132 I4132 P212121 P212121 Cell dimensions a, b, c (Å) 183.28, 183.28, 183.28 183.04, 183.04, 183.04 182.47, 182.47, 182.47 46.26, 58.54, 121.51 45.97, 58.39, 120.37 α=β=γ (deg) 90.0 90.0 90.0 90.0 90.0 Wavelength (Å) 1.075 1.127 1.075 1.075 1.075 Resolution (Å)a 50–2.10 (2.14–2.10) 50–1.90 (1.93–1.90) 50–2.0 (2.03–2.0) 50–3.0 (3.11–3.0) 50–2.45 (2.49–2.45) Rsym 0.127 (0.457) 0.088 (0.416) 0.142 (0.461) 0.139 (0.641) 0.096 (0.82) I/σI 42.4 (12.1) 27.2 (6.4) 33.4 (12.2) 13.5 (2.4) 16.2 (2.3) Completeness (%) 100 (100) 100 (100) 100 (100) 99.7 (99.0) 99.6 (99.8) Redundancy 42.1 (40.4) 10.8 (10.8) 42.4 (41.8) 6.5 (6.1) 6.4 (6.6) Refinement Resolution (Å) 50.0–2.1 50.0–1.9 50.0–2.0 50.0–3.0 50.0–2.7 No. reflections 30 835 41 208 35 089 7013 9317 Rwork/Rfree 0.189/0.232 0.198/0.226 0.200/0.213 0.246/0.265 0.203/0.252 No. atoms Protein 2337/275 aa 2337/275 aa 2337/275 aa 2210/268 aa 2221/269 aa CoA — 48/1 Molecule 48/1 Molecule — — H4K16CoA 60/1 Molecule — — — — Water 240 197 174 — 31 B-factors Protein 21.71 20.18 28.41 72.40 36.15 CoA 26.31 25.05 32.22 — — Water 28.14 25.21 32.21 — 28.57 R.m.s.d. Bond lengths (Å) 0.008 0.008 0.007 0.006 0.009 Bond angles (deg) 1.167 1.093 1.145 1.158 1.176 a Values in parentheses are for the highest-resolution shell. A more detailed analysis of the electron density map proximal to the bisubstrate inhibitor in the active site revealed unmodelled density at the tip of the K262 side chain in the active site, located in the 14-residue α2–β7 loop. We modelled an acetyl group in this position and subsequent crystallographic refinement and visualization of a simulated annealing omit map reinforced the presence of acetylated K262 (K262Ac) (Figure 1B). To confirm that the observed acetylation of K262 was not an artefact of cocrystallizing Esa1 bound to the H4K16Ac bisubstrate inhibitor, we determined the crystal structure of Esa1 bound to CoA to 1.9 Å resolution (Table I; Supplementary Figure S1A) and again observed electron density consistent with acetylated K262 (Supplementary Figure S1A). In addition, we also observed well-formed electron density in place of the lysine portion of the bisubstrate inhibitor that we have modelled and refined as a cacodylate buffer molecule containing disordered oxygen ligands as observed in other structures (Raman et al, 2001) (Supplementary Figure S1A). Inspection of the environment proximal to K262Ac reveals that the acetyl group is buried in the active site of the enzyme, with the methyl group making van der Waals interactions with phenylalanine residues 271 and 273 of β7 and the carbonyl moiety hydrogen bonding to Y289 and S291 of β8 of the structurally conserved HAT core region (Figure 1B). Correlating with the significance of these interactions and its generality to other MYST proteins, K262, F271, F273, Y289 and S291 are highly conserved throughout the MYST family of proteins (Figure 1C). The acetyl group of K262Ac is also ∼7 Å from the sulphur atom of the bisubstrate inhibitor and ∼5 Å and 8 Å from the C304 and E338 catalytic residues, respectively (Figure 1B). Structure of hMOF reveals a conformational change upon active site acetylation To establish if analogous lysine acetylation might extend to MYST proteins in other species, we determined the crystal structure of hMOF (Figure 1A; Table I). Inspection of the electron density map proximal to K274 in hMOF, the analogous lysine to yEsa1 K262, revealed that, the α2–β7 loop bearing K274 adopts two conformations. In one of these conformations, K274 is acetylated and is oriented towards the active site of the enzyme, interacting with residues that are conserved and making analogous interactions with yEsa1 (F283, F285, Y301 and S303 in hMOF) (Figure 1D). In the second α2–β7 loop conformation, K274 is not acetylated and flipped out of the active site by ∼90° and pointing across the cognate protein lysine binding site (Figure 1D) to make a long H-bond (3.4 Å) with the E350 catalytic base residue located on the α3–α4 loop that flanks the opposite side of the cognate protein lysine binding site. A superposition of the hMOF and yEsa1/H4K16CoA structures (Figure 1E) also reveals that the α2–β7 loop has a more varied conformation than other protein elements surrounding the cognate protein lysine binding site and that the position of the unacetylated K274 of hMOF overlaps with the backbone of the lysine portion of the H4K16CoA bisubstrate inhibitor of yEsa1. These structural observations suggest that the unacetylated lysine adopts a conformation that inhibits cognate protein lysine acetylation, possibly by directly blocking substrate binding. To further confirm the presence of K262Ac and K274Ac in yEsa1 and hMOF, respectively, we subjected both recombinant proteins to LC–MS/MS and unambiguously identified the presence of peptides consistent with acetylation at these positions (Figure 1F; Table II). A weak MS signal for the unacetylated LFLDHK peptide was detected, indicating a low level of unacetylated K274 in wild-type (WT) hMOF (Table II). These results, together with the conservation of this lysine throughout the MYST family, suggest that analogous lysine acetylation occurs throughout this family of HAT proteins. Table 2. Mass spectrometry analysis of recombinant yEsa1 and hMOF proteinsa Active site acetylation by MYST proteins occurs by autoacetylation To demonstrate that the acetylation of recombinant yEsa1 and hMOF proteins was mediated by autoacetylation, we prepared the recombinant yEsa1 and hMOF HAT domains in bacteria and then treated them with the relatively promiscuous yeast Hst2 deacetylase plus NAD+ cofactor to deacetylate them. We then added nicotinamide to inhibit Hst2 activity and Ac-CoA to provide a cofactor for any potential autoacetylation activity. We subjected both the untreated and Ac-CoA treated HAT domains to LC–MS/MS to survey for acetylated and unacetylated peptides containing K262 and K274 of yEsa1 and hMOF, respectively. As shown in Table II, these studies revealed that Hst2-mediated deacetylation of the recombinant yEsa1 and hMOF samples reduced the acetylation levels of K262 and K274 to <1% and 10%, respectively, based upon normalized integrated areas of the acetylated tryptic peptides. Concurrently, a strong signal for the unacetylated smaller tryptic peptide was observed in these samples. Reacetylation upon Ac-CoA addition increased the extent of acetylation as indicated by the marked reduction of the unacetylated peptide for both proteins. These data are consistent with the conclusion that active site acetylation of yEsa1, hMOF, and likely other MYST proteins occurs by autoacetylation. To further characterize whether autoacetylation occurs in cis (intramolecularly) or trans (intermolecularly), we subjected the Hst2-treated yEsa1 to reacetylation with saturating concentrations of Ac-CoA and increasing concentrations of yEsa1 and measured the amount of autoacetylated yEsa1 using filter binding and scintillation counting (Yan et al, 2002). As can be seen in Figure 2A, these studies reveal that the rate of yEsa1 autoacetylation is largely first order with respect to protein concentration consistent with autoacetylation in cis. Autoacetylation in cis is also consistent with the structural observation that the autoacetylated lysine is located within the enzyme active site and about 5 Å away from the catalytic cysteine residue (C304 in yEsa1 and C316 in hMOF) that would transfer the acetyl group via a ping-pong catalytic mechanism (Yan et al, 2002) and about 7 Å from the CoA sulphur atom that might transfer the acetyl group via a ternary complex mechanism (Berndsen et al, 2007). Therefore, a relatively minor movement of the acetylated lysine side chain (e.g. a change of rotamer) and/or acetyl donors could easily accommodate autoacetylation in cis. Figure 2.Activity of MYST proteins harbouring mutation of the conserved acetyllysine residue. (A) Autoacetylation of the yEsa1 HAT domain as a function of protein concentration. Each data point was obtained in triplicate with standard deviations indicated with error bars. (B) Catalytic activities of the yEsa1, hMOF HAT domains and the ySas2/4/5 complex and of their corresponding KAc-R mutants. Activity is indicated along the ordinate as the radioactivity (in CPM) incorporated into a 21-residue N-terminal histone H4 peptide from [14C] Ac-CoA as a function of protein (along the abscissa). (C) Histone peptide quenching of tryptophan fluorescence of hMOF-wt and the hMOF-K274R and hMOF-K274A mutants. The fluorescence emission spectra with 0 or 7 μM of H4p21 peptide are shown. The insert shows the quenching percentage as a function of H4p21 peptide concentration. F is the fluorescence intensity of hMOF with H4p21 peptide and F0 is the fluorescence intensity of hMOF in the absence of peptide. Download figure Download PowerPoint Previous studies on MYST proteins revealed that a conserved glutamate residue (E338 in yEsa1 and E350 in hMOF) functions as a general base for cognate protein lysine substrate acetylation and mutation of this residue to glutamine results in near background levels of cognate protein acetyltransferase activity (Yan et al, 2000). In order to test if autoacetylation also requires this conserved glutamate residue, we prepared the HAT domains of yEsa1 and hMOF harbouring glutamate-to-glutamine mutations in these residues for crystal structure determination. The structure of yEsa1-E338Q revealed that K262 was still fully acetylated (Table I; Supplementary Figure S1B), and the structure of hMOF-E350Q revealed that the α2–β7 loop still adopted the same two conformations containing the acetylated and unacetylated K274 forms of the WT enzyme (Table I; Supplementary Figure S1C). This was also confirmed by LC–MS/MS mass spectrometry (Supplementary Figure S1D; Table II). These structural findings suggest that autoacetylation of the MYST proteins do not require, or are less dependent on, the conserved glutamate general base residue that is required for cognate protein lysine substrate acetylation. To more directly establish the dependence of MYST autoacetylation on the glutamate general base residue, we determined rate curves as a function of Ac-CoA concentration for the deacetylated yEsa1-wt and yEsa1-E338Q proteins (Supplementary Figure S1E). As can be seen from these rate curves, relative to yEsa1-wt, the yEsa1-E338Q mutant still showed appreciable activity with a reduction in Vmax of less than two-fold and an elevation in Km of less than five-fold for autoacetylation. This is in contrast to the effect of the E338Q mutation on cognate protein lysine acetylation, which shows near background levels of acetylation (Yan et al, 2000). These data demonstrate that MYST autoacetylation is significantly less dependent on the conserved glutamate general base residue than MYST cognate protein lysine acetylation. Autoacetylation is required for HAT activity and histone substrate binding in vitro To directly test the hypothesis that acetylation of K262 and K274 in yEsa1 and hMOF, respectively, contributes to cognate protein lysine acetylation, we prepared recombinant HAT domains containing the corresponding arginine mutations (yEsa1-K262R and hMOF-K274R) and measured their catalytic activities towards a histone H4 tail peptide substrate. These mutant proteins eluted from gel filtration at the same position as the WT protein (Supplementary Figure S2A and B), suggesting that they were properly folded. Interestingly, enzymatic analysis of these mutant proteins showed tha
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