Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation
2011; Springer Nature; Volume: 30; Issue: 14 Linguagem: Inglês
10.1038/emboj.2011.193
ISSN1460-2075
AutoresChuanbing Bian, Chao Xu, Jianbin Ruan, KK Lee, Tara L. Burke, W. Tempel, Dalia Barsyte, Jing Li, Minhao Wu, Bo Zhou, Brian Fleharty, Ariel Paulson, Abdellah Allali‐Hassani, Jin-Qiu Zhou, Georges Mer, Patrick A. Grant, Jerry L. Workman, Jianye Zang, Jinrong Min,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle17 June 2011free access Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation Chuanbing Bian Chuanbing Bian School of Life Sciences, University of Science and Technology of China, Anhui, People's Republic of China Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Chao Xu Chao Xu Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Jianbin Ruan Jianbin Ruan School of Life Sciences, University of Science and Technology of China, Anhui, People's Republic of China Search for more papers by this author Kenneth K Lee Kenneth K Lee Stowers Institute for Medical Research, Kansas City, MO, USA Search for more papers by this author Tara L Burke Tara L Burke Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, USA Search for more papers by this author Wolfram Tempel Wolfram Tempel Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Dalia Barsyte Dalia Barsyte Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Jing Li Jing Li School of Life Sciences, University of Science and Technology of China, Anhui, People's Republic of China Search for more papers by this author Minhao Wu Minhao Wu School of Life Sciences, University of Science and Technology of China, Anhui, People's Republic of China Search for more papers by this author Bo O Zhou Bo O Zhou The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Graduate School, Chinese Academy of Sciences, Shanghai, People's Republic of China Search for more papers by this author Brian E Fleharty Brian E Fleharty Stowers Institute for Medical Research, Kansas City, MO, USA Search for more papers by this author Ariel Paulson Ariel Paulson Stowers Institute for Medical Research, Kansas City, MO, USA Search for more papers by this author Abdellah Allali-Hassani Abdellah Allali-Hassani Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Jin-Qiu Zhou Jin-Qiu Zhou The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Graduate School, Chinese Academy of Sciences, Shanghai, People's Republic of China Search for more papers by this author Georges Mer Georges Mer Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Patrick A Grant Patrick A Grant Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, USA Search for more papers by this author Jerry L Workman Corresponding Author Jerry L Workman Stowers Institute for Medical Research, Kansas City, MO, USA Search for more papers by this author Jianye Zang Corresponding Author Jianye Zang School of Life Sciences, University of Science and Technology of China, Anhui, People's Republic of China Search for more papers by this author Jinrong Min Corresponding Author Jinrong Min Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Department of Physiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Chuanbing Bian Chuanbing Bian School of Life Sciences, University of Science and Technology of China, Anhui, People's Republic of China Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Chao Xu Chao Xu Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Jianbin Ruan Jianbin Ruan School of Life Sciences, University of Science and Technology of China, Anhui, People's Republic of China Search for more papers by this author Kenneth K Lee Kenneth K Lee Stowers Institute for Medical Research, Kansas City, MO, USA Search for more papers by this author Tara L Burke Tara L Burke Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, USA Search for more papers by this author Wolfram Tempel Wolfram Tempel Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Dalia Barsyte Dalia Barsyte Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Jing Li Jing Li School of Life Sciences, University of Science and Technology of China, Anhui, People's Republic of China Search for more papers by this author Minhao Wu Minhao Wu School of Life Sciences, University of Science and Technology of China, Anhui, People's Republic of China Search for more papers by this author Bo O Zhou Bo O Zhou The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Graduate School, Chinese Academy of Sciences, Shanghai, People's Republic of China Search for more papers by this author Brian E Fleharty Brian E Fleharty Stowers Institute for Medical Research, Kansas City, MO, USA Search for more papers by this author Ariel Paulson Ariel Paulson Stowers Institute for Medical Research, Kansas City, MO, USA Search for more papers by this author Abdellah Allali-Hassani Abdellah Allali-Hassani Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Jin-Qiu Zhou Jin-Qiu Zhou The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Graduate School, Chinese Academy of Sciences, Shanghai, People's Republic of China Search for more papers by this author Georges Mer Georges Mer Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Patrick A Grant Patrick A Grant Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, USA Search for more papers by this author Jerry L Workman Corresponding Author Jerry L Workman Stowers Institute for Medical Research, Kansas City, MO, USA Search for more papers by this author Jianye Zang Corresponding Author Jianye Zang School of Life Sciences, University of Science and Technology of China, Anhui, People's Republic of China Search for more papers by this author Jinrong Min Corresponding Author Jinrong Min Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Department of Physiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Author Information Chuanbing Bian1,2,‡, Chao Xu2,‡, Jianbin Ruan1,‡, Kenneth K Lee3,‡, Tara L Burke4,‡, Wolfram Tempel2, Dalia Barsyte2, Jing Li1, Minhao Wu1, Bo O Zhou5, Brian E Fleharty3, Ariel Paulson3, Abdellah Allali-Hassani2, Jin-Qiu Zhou5, Georges Mer6, Patrick A Grant4, Jerry L Workman 3, Jianye Zang 1 and Jinrong Min 2,7 1School of Life Sciences, University of Science and Technology of China, Anhui, People's Republic of China 2Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada 3Stowers Institute for Medical Research, Kansas City, MO, USA 4Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, USA 5The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Graduate School, Chinese Academy of Sciences, Shanghai, People's Republic of China 6Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA 7Department of Physiology, University of Toronto, Toronto, Ontario, Canada ‡These authors contributed equally to this work *Corresponding authors: Stowers Institute for Medical Research, 1000 E. 50th Street Kansas City, MO 64110, USA. Tel.: +1 816 926 4392; Fax: +1 816 926 4692; E-mail: [email protected] of life sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027, People's Republic of China. Tel.: +86 551 3603433; Fax: +86 551 3600374; E-mail: [email protected] Genomics Consortium, University of Toronto, 101 College Street, MaRs Tower, Rm 736, Toronto, Ontario, Canada M5G 1L7. Tel.: +1 416 946 3868; Fax: +1 416 946 0588; E-mail: [email protected] The EMBO Journal (2011)30:2829-2842https://doi.org/10.1038/emboj.2011.193 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 SAGA (Spt–Ada–Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation. Introduction The SAGA (Spt–Ada–Gcn5 acetyltransferase) complex has been extensively investigated due to its important role in the regulation of gene expression (Baker and Grant, 2007; Rodriguez-Navarro, 2009). SAGA was identified in yeast as a 1.8-MDa histone acetyltransferase complex (Grant et al, 1997). The complex is highly conserved from yeast to humans, further confirming its importance in transcriptional regulation. Structural integrity of the complex requires three core components, Spt7, Spt20 and Ada1 (Wu and Winston, 2002). The SAGA complex contains two catalytic proteins, Gcn5 and Ubp8. Gcn5 is the histone acetyltransferase catalytic component, while Ubp8 is the histone deubiquitinating component (Rodriguez-Navarro, 2009). The complex contains about 20 proteins. In addition to the catalytic proteins of SAGA, Gcn5 and Ubp8, the remainder of the components have been shown to be important facilitators of either Upb8 or Gcn5 catalytic function. These other proteins modulate SAGA catalytic activities through their functional domains (Lee and Workman, 2007). These include an acetyllysine-binding bromodomain in Gcn5 and Spt7 (Zeng and Zhou, 2002); DNA-binding SWIRM domain in Ada2 (Qian et al, 2005; Da et al, 2006); and DNA- or histone-binding SANT domain in Ada2 (Boyer et al, 2004), and WD40 domains of unknown function in Taf5 (Durso et al, 2001) and Spt8 (Sermwittayawong and Tan, 2006) (Supplementary Figure S1). In 2002, Sgf29 was identified as a new component of the yeast SAGA complex (Sanders et al, 2002), but very little is known about its function. Like most SAGA proteins, Sgf29 is conserved from yeast to humans, and contains an N-terminal coiled-coil domain and C-terminal putative Tudor domains. More recently, the rat orthologue of Sgf29 was shown to directly bind Ada3, a SAGA component and important modulator of Gcn5 activity, via its N-terminal coiled-coil domain and activate c-Myc targeted gene expression (Kurabe et al, 2007). Downregulation of Sgf29 was able to suppress genes involved in c-Myc-mediated malignant transformation, implicating the important function of Sgf29 in proper gene regulation of STAGA, the mammalian homologue of SAGA. Most recently, a study by Vermeulen et al (2010) used mass spectrometry-based technologies to screen for interactors of activating and repressive tri-methyl marks on H3 and H4 in human cells. Sgf29 was shown to bind both H3K4me3 and H3K4me2 sites, with a slight preference for H3K4me3. ChIP sequencing revealed the presence of Sgf29 at gene promoters, which overlapped with the H3K4me3 mark. Conventional biochemistry methods confirmed the H3K4me2/3-specific binding by Sgf29. Sgf29, via its Tudor domains, was shown to be responsible for linking the human SAGA complex to H3K4me3 since knockdown of Sgf29 resulted in loss of H3K4me3 binding (Vermeulen et al, 2010). Therefore, Sgf29 acts as a molecular link between human SAGA and the post-translational modification H3K4me3. Many chromatin-associated proteins or protein complexes have both histone writing and reading activities, which are crucial for their proper function. For example, Clr4, the methyltransferase of the ClrC complex in fission yeast, contains both a catalytic SET domain and a chromodomain, which methylates histone H3K9 and binds H3K9me3, respectively (Min et al, 2002; Zhang et al, 2008). The ability of Clr4 to both write and read H3K9me is essential to spread heterochromatin and facilitate heterochromatin maintenance (Zhang et al, 2008). Sgf29, as a reader of H3K4 methyl marks for the SAGA complex, joins the group of other proteins in both yeast and humans known to recognize methyl marks for their respective HAT complex and target its proper acetylation. For example, Yng1 is known to target NuA3 HAT activity to the 5′ ORF in yeast by binding tri-methylated H3K4 via its PHD finger (Taverna et al, 2006). Similarly, in human cells, the PHD finger of the ING4 protein, a subunit in the HBO1 acetyltransferase complex, binds to tri-methylated H3K4, increasing acetylation by the HBO1 acetyltransferase at the promoter of target genes, leading to efficient transcription (Hung et al, 2009; Saksouk et al, 2009). Misregulation of these targeted processes can have costly outcomes when they control genes that require tight regulation. Such is the case with ING4, a known tumour suppressor, where it drives expression of genes involved in anti-cancer activities (Hung et al, 2009). Therefore, these subunit reader domains of HAT complexes are important to properly target writer functions, specifically acetyltransferase activity in this case, and misregulation or loss of their function can lead to aberrations in chromatin dynamics. In this work, our structural studies show that Sgf29 contains unique tandem Tudor domains at its C-terminus and our binding assays show that these tandem Tudor domains selectively bind H3K4me2/3. To elucidate the molecular mechanism of this specific recognition by Sgf29, we determined the crystal structures of the tandem Tudor domains of human and Saccharomyces cerevisiae Sgf29 in complex with different modified histone H3K4 peptides. Furthermore, our in vivo functional assays show that Sgf29 is required for histone H3 acetylation by the SAGA complex. Results and discussion Sgf29 preferentially recognizes histone H3K4me2/3 via its tandem Tudor domains Based on secondary structure prediction, we found that Sgf29 contains a coiled-coil domain at its N-terminus and putative tandem Tudor domains at its C-terminus (Figure 1A). In contrast to the sequence diversity at its N-terminus, we found that the C-terminal region of Sgf29 has relatively higher sequence identity than the N-terminus, especially within the conserved Tudor domains (Figure 1B). Figure 1.Crystal structures of human and yeast Sgf29 tandem Tudor domains. (A) Domain structures of budding yeast Sgf29 (Sc) and human SGF29 (Hs). The coiled-coil domain is coloured in orange, and the two Tudor domains are coloured in blue and green, respectively. The starting and ending residues of each domain are numbered. (B) Structure-based sequence alignment of Sgf29 homologues. The two conserved Tudor domains are framed and coloured in blue and green, respectively. The secondary structure elements of scSgf29 and hsSGF29 are indicated above and below the sequence alignment, respectively. H3K4me and H3A1 binding residues are numbered and marked by stars and dots, respectively. The sequence identity of the full-length and the tandem Tudor domains of yeast Sgf29 with the other three homologues are indicated beside the first and the third row, respectively. The alignment was created with Espript (http://espript.ibcp.fr/ESPript/ESPript/). Sc, S. cerevisiae; Sp, Schizosaccharomyces pombe; Dm, Drosophila melanogaster; Hs, Homo sapiens. β and η represent β strands and 310 helix, respectively. (C, D) Cartoon representation of the crystal structures of yeast Sgf29 and human SGF29 tandem Tudor domains, respectively. The two Tudor domains are coloured in blue and green, respectively, and the secondary structure regions in both proteins are marked. Download figure Download PowerPoint The Tudor domain, as an important member of the 'Royal Family' of histone-binding modules, is structurally similar to the chromo, PWWP and MBT domains (Maurer-Stroh et al, 2003), and has been shown to bind methylated histones (Adams-Cioaba and Min, 2009). Thus, it was compelling to speculate that Sgf29 may preserve this histone methyllysine binding ability. To better understand the binding specificity of human hsSGF29 and its yeast orthologue scSgf29, we used isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and fluorescence polarization (FP) assays to measure the binding affinity of both hsSGF29 and scSgf29 for histone H3K4, H3K9, H3K27, H3K36, H3K79 and H4K20 peptides bearing different methylation states. We found that both hsSGF29 and scSgf29 do not exhibit detectable binding to any of the H3K27, H3K36, H3K79 and H4K20 peptides, regardless of their methylation states (Table I). Instead, both Sgf29 proteins show strong binding to methylated H3K4 peptides and preferentially bind H3K4me2 and H3K4me3 marks (Table I). Yeast scSgf29 shows no detectable binding to the unmodified H3K4 peptide. Human hsSGF29 can still bind unmodified H3K4 peptide, but with nearly 50-fold weaker affinity (Kd=24.0 μM). Interestingly, hsSGF29 can also bind to H3K9me3 peptides as strongly as to the unmodified H3K4 peptide (Table I). Because the H3K9 peptides we used share the first 11 residues with the H3K4 peptides, and scSgf29 does not bind H3K9me3 peptide similar to its inability to bind the unmodified H3K4 peptide (Table I), presumably hsSGF29 binds H3K9 peptides via the K4 containing sequence. In order to corroborate this, we synthesized a peptide H36−20K9me3 covering residues 6–20 of histone H3. The length of this peptide is the same as the wild-type (WT) H3K9me3 peptide, and the residue K9me3 is in a position corresponding to the K4me3 residue from the N-terminus of H3. Our ITC-binding assay shows that the binding to hsSGF29 is completely abolished for the H36−20K9me3 peptide (Table I). Hence, the tandem Tudor domains of Sgf29 selectively recognizes H3K4me2/3 marks. Table 1. Binding affinities of human and yeast Sgf29 to different histone peptides Peptide name Kd (μM): hsSGF29 Kd (μM): scSgf29 ITC FP SPR FP H31–11K4me0 24±2 92±19 NBa NB H31–11K4me1 4±1 21±4 9.2±0.4 32±7 H31–11K4me2 1±0.1 4±1 1.1±0.1 5±2 H31–11K4me3 0.5±0.2 2±0.3 0.8±0.2 4±1 (2.3±0.3b) H31–15K9me3 20±1 NDa ND NB H319–33K27me0/1/2/3 ND NB ND NB H333–41K36me0/1/2/3 ND NB ND NB H371–85K79me0/1/2/3 ND NB ND NB H413–27K20me0/1/2/3 ND NB ND NB H36–20K9me3 NB ND ND ND H32–11K4me3 34±3 ND ND ND α-N-Ac-H31–13K4me3Rme2s NB ND ND ND H31–13K4me3Rme2s 0.5±0.1 ND ND ND a NB, no detectable binding by ITC, SPR or FP assays; ND, not determined. b The binding affinity of scSgf29 to H3K4me3 is measured by ITC in comparison with the Kd values obtained by SPR and FP. Sgf29 contains unique tandem Tudor domains To uncover the molecular architecture of the putative tandem Tudor domains of Sgf29, we determined the crystal structures of human hsSGF29 (residues 129–293) and S. cerevisiae scSgf29 (residues 113–259). The crystal structures show that both human and yeast Sgf29 indeed contain tandem Tudor domains at their C-termini. The scSgf29 and hsSGF29 structures are very conserved with an RMSD of 1.6 Å for all aligned Cα atoms, although scSgf29 and hsSGF29 only have 20% amino-acid sequence identity (Figure 1B). Each Tudor domain consists of five twisted anti-parallel β strands forming a typical barrel-like fold (Figure 1C and D). scSgf29 was crystallized with a maltose-binding protein (MBP) tag fused to aid crystallization (Supplementary Figure S2A–C). scSgf29 in complex with the methylated H3K4 peptides were crystallized at pH 4.0. At such low pH, scSgf29 can still bind H3K4me2/3, although the binding affinity decreased dramatically (Supplementary Figure S2D and E). The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face, which is distinct from other known tandem Tudor domain structures (Botuyan et al, 2006; Huang et al, 2006; Adams-Cioaba et al, 2010), which we will discuss below. Structural basis for the selective binding of Sgf29 to histone H3K4me2/3 peptides To shed light on the molecular mechanism of selective binding of Sgf29 to methylated histone H3K4, we determined the crystal structures of hsSGF29 (residues 115–293) and scSgf29 (residues 113–259) in complex with di- and tri-methylated H3K4 peptides, respectively. The structures of the H3K4me2–Sgf29 and H3K4me3–Sgf29 complexes are almost identical for both hsSGF29 and scSgf29 (Figure 2; Supplementary Figures S3 and S4). We used a longer hsSGF29 construct for crystallization of the complexes because crystals were of higher quality than those of the short construct (residues 129–291). The longer construct contains an extra α helix in the N-terminus, which is located between the two Tudor domains and sits outside the histone binding cleft (Figure 2B). Hence, this extra N-terminal α helix is not directly involved in histone binding, which is also confirmed by our binding results. Our studies show that the short hsSGF29 fragment binds as tightly as the long hsSGF29 fragment to the H3K4me3 histone peptide (Supplementary Figure S5). Figure 2.Selective binding of H3K4me2/3 by the SGF29 tandem Tudor domains. Interactions between yeast (A) and human SGF29 (B) tandem Tudor domains and H3K4me3 peptide. The Sgf29 tandem Tudor domains are shown in cartoon representation and coloured in blue. The H3K4me3 peptide is shown as a stick model. The conserved and non-conserved hydrogen bonds are coloured in red and black, respectively. (C, D) Electrostatic surface representation of yeast (C) and human (D) Sgf29–H3K4me3 complex. Sgf29 is shown in surface representation, and histone peptide is shown in a stick model. Download figure Download PowerPoint From the complex structures of both scSgf29 and hsSGF29, we can see that the first four residues of the H3K4me3 peptide are snugly embedded between the two Tudor domains (Figure 2A and B). The first residue in the H3K4me3 peptide (H3A1) is anchored in a small negatively charged pocket created by the first Tudor domain in both hsSGF29 and scSgf29 (Figure 2). The backbone amine group of H3A1 forms a conserved salt bridge with the carboxylate oxygen of D194 in hsSGF29 (D163 in scSgf29) (Figure 2). The importance of this H3A1-binding pocket is exemplified by the significant reduction in binding affinity (∼70-fold weaker) when the H3A1 residue is deleted from the H3K4me3 peptide (Table I). Because the H3A1-binding pocket is negatively charged and rigidly formed, acetylation of H3A1 completely abolishes H3K4me3 binding to hsSGF29 (Table I). Hence, the short side chain H3A1 residue is tightly secured in the small negatively charged pocket formed in the first Tudor domain of Sgf29. The tri-methyllysine K4me3 is bound in a conserved negatively charged pocket located in the second Tudor domain in both hsSGF29 and scSgf29, and is flanked by two aromatic residues (Y238 and Y245 in hsSGF29, and Y205 and Y212 in scSgf29) (Figure 2). The backbone amine group of the K4me3 also forms a hydrogen bond with the carbonyl oxygen of Y245 in hsSGF29 (Y212 in scSgf29). A third aromatic residue, F264 in hsSGF29 (F229 in scSgf29), lies underneath and buttresses Y238 (Y205 in scSgf29), although it also provides additional hydrophobic interaction with the methyllysine. A negatively charged residue D266 (E232 in scSgf29) on the other side of the K4me3-binding pocket interacts with the methyllysine via a salt bridge. Therefore, the tri-methyllysine residue K4me3 is anchored by the second Tudor domain through cation-π, van der Waals, hydrophobic, electrostatic and hydrogen bond interactions. We performed a series of mutagenesis experiments in hsSGF29 in order to verify the importance of the K4me3 and H3A1 binding residues. Mutating Y245 to alanine completely abolished the binding, mutating F264, Y238 or D266 to alanine markedly reduced the binding affinity (Table II), which is consistent with our structural observations that Y245 are essential in binding methylated H3K4me3, and Y238, F264 and D266 reinforce the binding. Table 2. Binding affinities of hsSGF29 and its mutants to histone H3K4me3 peptide (ARTKme3QTARKST) hsSGF29 wt and mutants Kd (μM) Wild type 2.2±0.3 D194A NB D194R NB D196A 28±2 D196R NB D194A_D196A NB Y238A 123±13 Q240A 5.5±0.7 T242A 832±207 Y245A NB F264A 128±16 D266A 104±18 NB, no detectable binding. Mutating D194 to alanine or D194/D196 to alanines in hsSGF29 disrupts binding to H3K4me3 (Table II), underlying the importance of D194 in H3K4m2/3 binding through anchoring the H3A1 residue. The D196A mutant could still bind H3K4me3 although with an ∼12-fold weaker affinity (Table II). In human but not yeast Sgf29, D196 forms an extra hydrogen bond with the H3A1 residue. D196 is replaced by E165 in scSgf29, which does not form the hydrogen bond with H3A1 (Figure 2A). D196R mutation completely disrupts binding to H3K4me3 (Table II), which indicates that the negative charge around the H3A1-binding pocket is necessary to stabilize the complex. The backbone of Arg2 in H3 (H3R2) forms two hydrogen bonds with the backbone amine group and the side chain carbonyl oxygen of Thr242 in hsSGF29. Mutating Thr242 to alanine severely diminishes the binding (Table II). Nevertheless, the H3R2 side chain is flexible and does not form any conserved interactions with hsSGF29 (Figure 2; Supplementary Figure S3D). This explains why methylation of H3R2 does not affect its binding to hsSGF29 (Kd=0.5 μM; Table I). Sgf29 has unique tandem Tudor domains, distinct from those of JMJD2A, FMR1, 53BP1 and SND1 So far, the crystal structures of a few tandem Tudor domains have become available. Based on the architecture of these Tudor domains, tandem Tudor domains can be classified into five subfamilies, i.e., Sgf29, JMJD2A (Huang et al, 2006), 53BP1 (Botuyan et al, 2006), FMR1 (Ramos et al, 2006; Adams-Cioaba et al, 2010) and SND1 (Liu et al, 2010a, 2010b) subfamilies. The two Tudor domains in Sgf29 face each other with the first two β strands from each Tudor domain packing against each other (Figure 3A and B), accordingly named as 'face-to-face' tandem Tudor domains. The two Tudor domains in 53BP1 line up one after the another with the first two β strands from the first Tudor packing against the last two β strands from the second Tudor (Figure 3C) (Botuyan et al, 2006), named as 'lineup' tandem Tudor domains. The two Tudor domains in the FMR1 subfamily of Tudor proteins, such as FMR1 (Ramos et al, 2006), FXR1/2 (Adams-Cioaba et al, 2010) and UHRF1, form a head-to-head architecture with the same ends of the β strands packing against each other (Figure 3D). JMJD2A has hybrid Tudor domains (Figure 3E) (Huang et al, 2006). The extended Tudor domain in SND1 and other TDRD members was predicted to contain a single Tudor domain, but the recent structural studies demonstrated that the N- and C-terminal extensions surrounding the canonical Tudor domain in SND1 and other TDRD members fold together and form a Tudor-like domain (Figure 3F) (Liu et al, 2010a, 2010b). All these five kinds of tandem Tudor domains bind lysine/arginine methylated proteins, but only in Sgf29 do both Tudor domains extensively bind methylated histones (Supplementary Figure S6). Two negatively charged pockets are formed in each individual Tudor domain, and the length between these two pockets determines the selectivity of Sgf29. The other four kinds of tandem Tudor domains mainly use one Tudor domain to interact with their corresponding ligands. That explains why they have less strict sequence selectivity. For example, JMJD2A cannot only bind H3K4me3, but also binds H4K20me3 in an opposite orientation (Lee et al, 2008). Figure 3.Architecture of different double Tudor domains. (A) Human SGF29. (B) Yeast Sgf29. (C) 53BP1 (PDB code 2G3R). (D) FXR1 (PDB code 3O8V). (E) Hybrid Tudor domain protein JMJD2A (PDB code 2GFA). (F) The extended Tudor domain of SND1 (PDB code 3OMC). The Tudor domains are coloured in rainbow spectrum on the basis of topology, exc
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