Molecular recognition of histone lysine methylation by the Polycomb group repressor dSfmbt
2009; Springer Nature; Volume: 28; Issue: 13 Linguagem: Inglês
10.1038/emboj.2009.147
ISSN1460-2075
AutoresClemens Grimm, Raquel Matos, Nga Ly‐Hartig, Ulrich Steuerwald, Doris Lindner, Vladimir Rybin, Jürg Müller, Christoph W. Müller,
Tópico(s)RNA modifications and cancer
ResumoArticle4 June 2009Open Access Molecular recognition of histone lysine methylation by the Polycomb group repressor dSfmbt Clemens Grimm Clemens Grimm European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, FrancePresent address: Institut für Biochemie, Biozentrum der Universität, Würzburg, Am Hubland, D-97074 Würzburg, Germany Search for more papers by this author Raquel Matos Raquel Matos European Molecular Biology Laboratory, Gene Expression Unit, Heidelberg, Germany Search for more papers by this author Nga Ly-Hartig Nga Ly-Hartig European Molecular Biology Laboratory, Gene Expression Unit, Heidelberg, Germany Search for more papers by this author Ulrich Steuerwald Ulrich Steuerwald European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany Search for more papers by this author Doris Lindner Doris Lindner European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany Search for more papers by this author Vladimir Rybin Vladimir Rybin European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany Search for more papers by this author Jürg Müller Corresponding Author Jürg Müller European Molecular Biology Laboratory, Gene Expression Unit, Heidelberg, Germany Search for more papers by this author Christoph W Müller Corresponding Author Christoph W Müller European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany Search for more papers by this author Clemens Grimm Clemens Grimm European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, FrancePresent address: Institut für Biochemie, Biozentrum der Universität, Würzburg, Am Hubland, D-97074 Würzburg, Germany Search for more papers by this author Raquel Matos Raquel Matos European Molecular Biology Laboratory, Gene Expression Unit, Heidelberg, Germany Search for more papers by this author Nga Ly-Hartig Nga Ly-Hartig European Molecular Biology Laboratory, Gene Expression Unit, Heidelberg, Germany Search for more papers by this author Ulrich Steuerwald Ulrich Steuerwald European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany Search for more papers by this author Doris Lindner Doris Lindner European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany Search for more papers by this author Vladimir Rybin Vladimir Rybin European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany Search for more papers by this author Jürg Müller Corresponding Author Jürg Müller European Molecular Biology Laboratory, Gene Expression Unit, Heidelberg, Germany Search for more papers by this author Christoph W Müller Corresponding Author Christoph W Müller European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany Search for more papers by this author Author Information Clemens Grimm1, Raquel Matos2, Nga Ly-Hartig2, Ulrich Steuerwald3, Doris Lindner3, Vladimir Rybin3, Jürg Müller 2 and Christoph W Müller 3 1European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France 2European Molecular Biology Laboratory, Gene Expression Unit, Heidelberg, Germany 3European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany *Corresponding authors: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany. Tel.: +49 6221 387 8320; Fax: +49 6221 387 519; E-mail: [email protected] Molecular Biology Laboratory, Gene Expression Unit, 69117 Heidelberg, Germany. Tel.: +49 6221 387 629; Fax: +49 6221 387 518; E-mail: [email protected] The EMBO Journal (2009)28:1965-1977https://doi.org/10.1038/emboj.2009.147 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Polycomb group (PcG) proteins repress transcription by modifying chromatin structure in target genes. dSfmbt is a subunit of the Drosophila melanogaster PcG protein complex PhoRC and contains four malignant brain tumour (MBT) repeats involved in the recognition of various mono- and dimethylated histone peptides. Here, we present the crystal structure of the four-MBT-repeat domain of dSfmbt in complex with a mono-methylated histone H4 peptide. Only a single histone peptide binds to the four-MBT-repeat domain. Mutational analyses show high-affinity binding with low peptide sequence selectivity through combinatorial interaction of the methyl-lysine with an aromatic cage and positively charged flanking residues with the surrounding negatively charged surface of the fourth MBT repeat. dSfmbt directly interacts with the PcG protein Scm, a related MBT-repeat protein with similar methyl-lysine binding activity. dSfmbt and Scm co-occupy Polycomb response elements of target genes in Drosophila and they strongly synergize in the repression of these target genes, suggesting that the combined action of these two MBT proteins is crucial for Polycomb silencing. Introduction Polycomb group (PcG) proteins are transcriptional regulators required for the repression of developmental control genes in animals and plants. PcG proteins exist in distinct multi-protein complexes that repress transcription by modifying the chromatin of target genes and thereby generating transcriptional off states that can be stably and heritably maintained (Francis and Kingston, 2001; Schwartz and Pirrotta, 2007). To date, three principal PcG multi-protein complexes have been identified and characterized: Pho repressive complex (PhoRC), PRC2 and the two related complexes PRC1 and dRAF (Schwartz and Pirrotta, 2007; Muller and Verrijzer, 2009). Among those, the PhoRC subunit Pho is the only sequence-specific DNA-binding PcG protein. Studies in Drosophila showed that PcG complexes assemble at specific cis-regulatory sequences in target genes, called Polycomb response elements (PRE), and that PhoRC has a central function in providing a PRE-binding platform that allows for the assembly of the chromatin-binding PRC1 and PRC2 complexes (Wang et al, 2004; Mohd-Sarip et al, 2005; Klymenko et al, 2006). In addition to Pho, PhoRC contains dSfmbt (Klymenko et al, 2006). In Drosophila, dSfmbt, the PRC1 subunit Sex comb on midleg (Scm) and a third protein, called L(3)mbt, form a small protein family with a very similar and unique domain architecture. The central portion of each protein contains an MBT-repeat domain that consists of two (Scm), three (L(3)mbt) or four (dSfmbt) repeats, and each protein contains Zn-finger motifs in the N-terminus and a sterile alpha motif (SAM) domain at the very C-terminus. Studies on dSfmbt, first showed that MBT-repeat domains selectively bind to mono- and dimethylated lysine residues in histones, but that they show low specificity for any particular histone lysine (Klymenko et al, 2006). Recent studies reported the crystal structures of the MBT domains of Scm and L3MBTL1 in complex with methylated histone-tail peptides (Grimm et al, 2007; Li et al, 2007; Min et al, 2007; Santiveri et al, 2008). In both proteins, the mono- or dimethylated histone lysine residues bind to the second MBT repeat and the interactions between the methyl-lysine side chain and an aromatic pocket in this repeat contribute the major part of the binding energy, whereas histone residues adjacent to the methyl-lysine form few interactions (Grimm et al, 2007; Li et al, 2007; Min et al, 2007; Santiveri et al, 2008). Consistent with this mode of recognition, the MBT-repeat domain of Scm binds histone-tail peptides, mono-methylated at H3-K9 or H4-K20 with a low affinity of about 500–800 μM (Grimm et al, 2007; Santiveri et al, 2008), whereas for binding of L3MBTL1 to the same mono-methylated lysines in peptides, two studies reported different affinities ranging from 140 to 400 μM (Min et al, 2007) or from 5 to 10 μM (Li et al, 2007). Interestingly, two distinct MBT-repeat-containing proteins, Scm and dSfmbt, are both essential components of the PcG-repression system in Drosophila. Functional studies on Scm showed that mutations in the MBT-repeat domain that abolish methyl-lysine binding in vitro impede the Polycomb-repressor function of this protein in Drosophila (Grimm et al, 2007). Intriguingly, dSfmbt binds the same methylated lysines in histones bound by Scm but with about 100-fold higher affinity than Scm (Klymenko et al, 2006; Grimm et al, 2007; Santiveri et al, 2008). These observations, together with the lack of knowledge of sequence-specific methyl-lysine recognition by the L3MBTL1 or Scm MBT-repeat domains prompted us to characterize the MBT-repeat domain of dSfmbt at the structural and functional level. Here, we report the crystal structure of the MBT-repeat domain of dSfmbt in complex with a histone H4 peptide, mono-methylated at lysine 20 (H4K20me1). Using isothermal calorimetry (ITC), we evaluate the binding specificity of dSfmbt for different histone-tail peptides methylated at particular lysine residues and assess the contribution of residues adjacent to the methyl-lysine residue by mutational analysis. Functional tests in Drosophila show that dSfmbt and Scm act in a highly synergistic manner to maintain repression at Polycomb target genes in vivo and suggest a role for the Scm–dSfmbt heterodimer in chromatin compaction. Results and discussion Overall structure of the four-MBT-repeat domain of dSfmbt The structure of the four-MBT-repeat domain of D. melanogaster dSfmbt (dSfmbt-4MBT, Mr=51 kDa, residues 535–977) was solved in complex with a histone H4 tail peptide centred onto H4K20me1 at 2.8 Å resolution (Table I). To favour crystallization, three point mutations (K715D, R886S and R900D) were introduced on the surface of the dSfmbt-4MBT construct; these mutations do not significantly affect H4K20me1 binding (Table II, Materials and Methods). The overall structure of the dSfmbt-4MBT–peptide complex is shown in Figure 1. As in Scm and L3MBTL1, each MBT repeat consists of a central five-stranded β-core and an elongated N-terminal arm that contacts the neighbouring repeat. Repeat 2, 3 and 4 form a propeller-like structure with three-fold pseudo-symmetry similar to L3MBTL1 (Wang et al, 2003). Repeat 1 is docked onto the outer rim of this propeller in the area of repeat 4 and forms most contacts with repeat 4 but also interacts with the adjacent repeat 2 through the N-terminal arm of this repeat. The arm of repeat 1 forms most of the contact surface to repeat 4 and its conformation is therefore less extended compared with the three other arms (Figure 1B). The combination of these interactions between the four repeats thus results in a compact MBT-repeat domain. Figure 1.Structure of the four MBT-repeat domain of dSfmbt. (A) Ribbon diagram of the four MBT repeats of dSfmbt coloured in blue (repeat 1), green (repeat 2), yellow (repeat 3) and red (repeat 4). Histone H4K20me1 peptide is shown in grey. (B) Superposition of the core folds (grey) of MBT repeats 1–4. For each repeat, helix α2 and the arm regions are coloured according to (A). (C) dSfmbt core domains of repeat 1–4 aligned with core domains of MBT repeats in Drosophila Scm and human L3MBTL1. Positions corresponding to cage-forming residues in dSfmbt repeat 4, Scm repeat 2 and L3MBTL1 repeat 2 are marked with a red box and conserved cage-forming residues are depicted in red. dSfmbt residues contacting the H4K20me1 peptide are indicated with an asterisk. dSfmbt, Scm and L3MBTL1 residues important for differential peptide binding are drawn on red, blue and green background (compare text). N-terminal arm regions are less well conserved and are not included in the alignment. Download figure Download PowerPoint Table 1. Crystallographic data collection, phasing and model refinement statistics Data statistics Native Hg derivativea Hg-λ1 Hg-λ2 Space group P22121 C222 Cell axes (Å) 70.8, 97.0, 214.1 100.4, 140.0, 275.2 Wavelength (Å) 0.9330 1.0085 1.0064 Resolution range (Å)b 30–2.8 (3.0–2.8) 30–3.2 (3.37–3.2) Reflections observed/unique 226 138/37 129 137 646/32 417 137 646/32 401 Completeness (%) 99.8 (99.9) 97.6 (91.4) 97.8 (91.7) / 12.3 (3.3) 4.4 (1.7) 4.4 (1.7) Rsym (%)c 13.7 (55.1) 8.5 (49.0) 8.2 (48.0) Resolution range (Å) 30–3.2 ano iso/ano RCullis (acentric) (%) 0.82 0.72/0.88 Phasing power (acentric) 0.99 0.96/0.88 Overall figure of merit 0.50 Resolution range (Å) 30–2.8 (2.87–2.8) Rcryst/Rfree(%)d 22.3 (32.6)/25.4 (36.0) Overall B-factor [Å2] 28.9 Protein main/side chain [Å2] 28.0/29.9 Peptide ligand [Å2] 34.7 r.m.s.d. bond lengths [Å] 0.009 r.m.s.d. angles (deg) 1.35 Residues in Ramachandran plot (%): Most favoured region 89.4 Allowed 9.9 Generously allowed 1.1 Disallowed 0.0 a Hg derivative with three sites per molecule was obtained by soaking crystals for 1 h with 0.25 mM EMTS. Hg-λ1 was used as reference data set in the two-wavelengths MAD experiment. b Values in parentheses refer to the outer resolution shell. c Rsym=∑∣I− ∣/∑I, where I is the observed intensity of a given reflection. d Rcryst=∑∣Fo−Fc∣/∑Fo, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. Rfree is equal to Rcryst for a randomly selected 5% subset of reflections not used in the refinement. Table 2. Binding affinity of dSfmbt-4MBT for methyl-lysine-containing histone peptides Wild-type dSfmbt-4MBTa KD (μM) Mutant dSfmbt-4MBT protein KD (μM) D697A D808A D917A E947A/Y948F E947A/Y948F/D917A H4K2012–27—KGGAKRHRK20VLRDNIQ-CONH2 H4K20 >1000 H4K20me1 1.1±0.1b (11±0.4) 1.6±0.1 2.9±0.1 >1000 1.9±0.1 (18±0.5) >1000 H4K20me2 2.8±0.2 2.7±0.3 >1000 H4K20me3 >1000 H4K2017–23—RHRK20VLR-CONH2 H4K20me1 (7-mer) 1.5±0.03 (12.8±1.0) H4K2018–22—HRK20VL-CONH2 H4K20me1 (5-mer) 23±1 H4K2019–21—RK20V-CONH2 H4K20me1 (3-mer) 40±2.4 H4K20-R19A12–27—KGGAKRHAK20VLRDNIQ-CONH2 H4K20me1-R19A 4.6±0.3 (60±4) H4K2012–27 (scrambled)—LNRQDIAGK20GKHVKRR-CONH2 2.8±0.1 (45±3) H3K41–15—ARTK4QTARKSTGGKA-CONH2 H3K4me1 2.8±0.1 H3K4me2 2.2±0.1 H3K91–15—ARTKQTARK9STGGKA-CONH2 H3K9 85±3 H3K9me1 4.7±0.3 (21±0.4) 1.9±0.1 6.7±0.4 340±80 H3K9me2 15.6±0.9 (36±4) H3K9me3 85±10 H3K98–10—RK9S-CONH2 H3K9me1 134±8 (125±6) H3K2719–35—QLATKAARK27SAPATGGV-CONH2 H3K27 40±12 H3K27me1 2.9±0.4 H3K27me2 12.0±1.0 H3K27me3 >1000 H3K3628–43—SAPATGGVK36KPHRYRPG-CONH2 H3K36me1 5.0±0.1 H3K36me2 3.4±0.1 H3K7972–86—REIAQDFK79TDLRFQS-CONH2 H3K79me1 >1000 H3K79me2 >1000 a Values in parentheses were measured at 150 mM NaCl. b KD=2.1±0.1 μM for dSfmbt construct K715D/R886S/R900D (residue 535–977) used for co-crystallization. H4K20me1 peptide binds to the fourth MBT repeat of dSfmbt In the complex structure, the H4K20me1 peptide (RHRKme1VLR) interacts with dSfmbt MBT repeat 4 (Figure 1A). Interactions between dSfmbt and the peptide are mediated by the central mono-methylated lysine, which points in the binding pocket on top of the β-barrel of the fourth MBT repeat (Figure 2) but also through a combination of polar and hydrophobic interactions of adjacent peptide residues with residues of repeat 4. Figure 2.Methyl-lysine peptide recognition by dSfmbt. Details of the bound histone H4K20me1 peptide binding to the aromatic cage pocket within MBT repeat 4. The simulated annealing omit electron-density map for the ligand is shown in wire–frame mode. Download figure Download PowerPoint The methyl-lysine-binding pocket of the fourth repeat is formed by residues Phe941, Trp944 and Tyr948, whose aromatic planes are oriented perpendicular to each other, forming roughly the corner of a cube. The methyl-lysine side chain closely packs against the aromatic side chains of Tyr948 and Trp944. Compared with the 'aromatic cage' in Scm (Grimm et al, 2007), we observe a significant distortion of the ideal rectangular geometry, mainly because dSfmbt-residue Tyr948 is oriented at an angle of approximately 60° with respect to Trp944. On the other side of the binding pocket, Asp917 binds the ε-amino group of H4K20me1 through a direct hydrogen bond assisted by electrostatic interactions. Furthermore, the pocket is outlined by residue Cys925. In addition to the interactions with the mono-methylated lysine, a salt bridge connects dSfmbt Glu947 (corresponding to Scm Ala354) with Arg19 in histone H4, whereas the hydroxyl group of Tyr948 (corresponding to Scm Phe355) forms a hydrogen bond with the Nε atom of this arginine (Figure 2). In the dSfmbt–peptide complex, electron density can be unambiguously assigned for six of the seven peptide residues (Figure 2). A peptide surface of 480 Å2 contacts dSfmbt, whereby 40% of the interaction surface is contributed by the mono-methylated lysine residue. Contributions of H4K20me1 and dSfmbt residues to the peptide-binding affinity We used ITC to evaluate binding of dSfmbt to methylated histone-tail peptides. First, we tested the binding of dSfmbt-4MBT to 16-residue peptides that were either unmodified, mono-, di- or tri-methylated at H4K20 (Table II, ITC profiles are depicted in Supplementary Figure S1). Mono- and dimethylated H4K20 peptides were bound with 1 and 3 μM affinity, respectively, whereas unmethylated and tri-methylated H4K20 peptides were bound with approximately 500-fold lower affinities (KD>1000 μM, Table II). To probe the contribution of residues flanking the methyl-lysine, we next tested binding to shorter H4K20me1 peptides. The heptameric peptide used for co-crystallization was bound with an affinity comparable to the 16-residue peptide. However, further shortening to a five-residue peptide reduced the affinity to 23 μM (Table II). This suggests that contributions provided by residues Arg17 and especially Arg23 that is well ordered in the crystal structure (Figure 2) are responsible for the approximately 15-fold higher affinity for the heptameric peptide. An even shorter three-residue peptide was bound with a KD value of 40 μM (Table II), indicating that His18 and Leu22, both pointing away from the MBT surface, contribute little to the binding affinity. The next residue Arg19 directly adjacent to K20me1 is involved in polar interactions with dSfmbt (Figure 2) and in the context of the 16-residue H4K20me1 peptide, mutating Arg19 into alanine reduces the binding affinity by about four-fold (Table II). In a complementary set of experiments, we mutated dSfmbt residues Glu947 and Tyr948 to generate a dSfmbtE947A/Y948F protein (Figure 1C). Compared with wild-type dSfmbt, the dSfmbtE947A/Y948F protein bound the 16-residue H4K20me1 peptide with similar affinity (Table II), presumably because the change from Tyr948 to Phe948 still permits the π−cation interaction with the guanidinium group of Arg19. However, mutating the methyl-lysine-contacting Asp917 into alanine in the single-mutant dSfmbtD917A or triple-mutant dSfmbtE947A/Y948F/D917A proteins completely abolished their ability to bind to H4K20me1 (Table II) without affecting the overall fold and thermal stability of the domain (data not shown). As control, we also tested alanine substitutions of the conserved Asp697 or Asp808 residues at the corresponding positions in the second or third repeat, respectively, (i.e. dSfmbtD697A and dSfmbtD808A) but found that these mutations did not significantly affect peptide binding (Table II). In summary, these results suggest that dSfmbt binds H4K20me1 with high affinity through the combined interaction of the MBT-binding pocket with the mono-methylated lysine and multiple contacts on the MBT surface with histone residues flanking the methyl-lysine. Binding of dSfmbt to other methylated histone peptides Despite the high selectivity of dSfmbt in discriminating between different lysine methylation states, it is able to recognize mono- and dimethylated lysine in a broad range of sequence contexts: dSfmbt also binds histone peptides mono- or dimethylated at H3K4, H3K9, H3K27 or H3K36 with affinities ranging between 1 and 16 μM (Table II). Furthermore, a scrambled H4K20me1 peptide is bound with similar affinity as the native H4K20me1 peptide but more negatively charged peptides such as mono- or dimethylated H3K79 peptides (pI 4.4) are bound with an affinity below 1000 μM (Table II). It thus seems that charge complementarity between the positively charged amino acids in histone-tail peptides (pI values 11–12) and the overall negatively charged dSfmbt surface (Figure 3) rather than recognition of individual residues outside the methyl-lysine-binding pocket is important for the interaction. Given the low sequence specificity, we currently cannot exclude that dSfmbt recognizes methyl-lysine residues in other proteins, although so far only interactions between MBT-repeat proteins and mono- and di-methyl-lysine-containing histone tails have been reported (Kim et al, 2006; Trojer et al, 2007; Wu et al, 2007). Figure 3.Comparison of the MBT-repeat-domain crystal structures of dSfmbt, L3MBTL1 and Scm. (A) Ribbon diagram of dSfmbt (left), L3MBTL1 (middle) and Scm (right). Equivalent MBT repeats as indicated by comparison of their tertiary structures are depicted with equivalent colours. (B) Electrostatic surface representation of dSfmbt, L3MBTL1 and Scm. The bound peptide ligands are depicted in yellow. In L3MBTL1, the methyl-lysine peptide is bound to MBT repeat 2. (C) Comparison of the surface conservation in dSfmbt, L3MBTL1 and Scm. Conserved regions with >50% sequence conservation are depicted in colour, dark green corresponds to strictly conserved residues. For surface comparison, orthologous sequences were aligned as depicted in Supplementary Figure S2. The following sequences were used for the alignments: dSfmbt: Drosophila melanogaster, Q9VK33, corresponds to the dSfmbt-4MBT crystal structure reported here; Anopheles gambiae, Q7Q0R1; Xenopus laevis, Q32N90; Mus musculus, P59178; Homo sapiens, Q05BQ5; Gallus gallus, Q5ZLC2; Tetraodon nigroviridis, Q4T9N5. L3MBTL1: Homo sapiens, Q9Y468, corresponds to the L3MBTL1 crystal structure (PDB accession code 2RHI); Bos tauru, Q08DF3; Gallus gallus, XP_417302; Danio rerio XP_699604. Scm: Drosophila melanogaster, Q9VHA0, as present in the Scm crystal structure (PDB accession code 2R57); Xenopus tropicalis, Q0IHT6; Homo sapiens, SCML2; Ciona intestinalis Q4H2U6. Download figure Download PowerPoint Previous binding studies using fluorescence polarization (FP) assays suggested more pronounced sequence selectivity for dSfmbt binding to H4K20me1/2 and H3K9me1/2 as opposed to binding to H3K4me1/2 or H3K27me1/2 (Klymenko et al, 2006). As our ITC measurements reported here provided little evidence for such binding selectivity, we repeated the binding assays with FP assays. To this end, we used a set of peptides that had been produced during the same synthesis reaction as those used for our ITC measurements but, in addition, had been modified by coupling fluorescent carboxylic acid to the N-terminus in the final synthesis step. In FP assays with these peptides, dSfmbt bound H4K20me1/2, H3K4me1/2, H3K9me1/2, H3K27me1/2 and H3K36me1/2 with comparably low micromolar affinities and the determined KD values were similar to those measured by ITC (Supplementary Table 1). The failure to detect high-affinity binding of dSfmbt to H3K4me1/2 or H3K27me1/2 by Klymenko et al (2006) might be because of differences in the method of peptide labelling (i.e. post-synthetic labelling) used in the previous study (W Fischle, personal communication). Taken together, ITC and FP assays reported here both gave comparable results and suggest that mono- and dimethylated lysines in the N-termini of H3 and H4 are all bound with similar micromolar affinities, whereas unmethylated and tri-methylated peptides are bound with much reduced affinity. Comparison of the dSfmbt, L3MBTL1 and Scm MBT-repeat domains The three MBT repeats of L3MBTL1 can be superimposed onto dSfmbt repeats 2, 3 and 4 (r.m.s.d.300Cα=6.1 Å, Z-score=22.6) using programme DALI (Holm and Sander, 1993), which identifies repeat 1 as the additional repeat in dSfmbt (Figure 3A). Interestingly, the N-terminal ends of the superimposed L3MBTL1 and dSfmbt structures lie in close vicinity, supporting the hypothesis that repeat 1 of dSfmbt was inserted during evolution. Scm MBT-repeats 1 and 2 can be superimposed onto repeats 1 and 3 of L3MBTL1 (r.m.s.d.200Cα=2.0 Å, Z-score=20.9) and repeats 2 and 4 of dSfmbt (r.m.s.d.193Cα=3.8 Å, Z-score=17.0). Therefore, repeat 2 of L3MBTL1 and the homologous repeat 3 of dSfmbt seem as extra features inserted between the two flanking MBT repeats of Scm (Figure 3A). The high r.m.s.d. between dSfmbt repeats 2–4 and L3MBTL1 repeats 1–3 mainly results from the more open arrangement of the three L3MBTL1 repeats, which are arranged around a central channel running along their three-fold pseudo-symmetry axis (Figure 3B). In the crystal structure, this channel is filled with solvent and bound sucrose molecules used as cryoprotectant. However, it could also serve as additional ligand-binding site as it is lined with conserved residues (Figure 3C). Scm binds mono-methyl-lysine-containing peptides with dissociation constants of approximately 500 μM (Grimm et al, 2007), whereas dSfmbt binds peptides with dissociation constants in the low micromolar range and up to 500 times better than Scm. These differences probably result from their differently charged surfaces (Figure 3B). In Scm, the methyl-lysine-binding pocket is lined by several basic residues (Lys326, Arg352 and His384, Figure 1C), which point towards the positively charged histone-tail peptide. In contrast, the corresponding dSfmbt residues (Met919, Thr945 and Pro976) are uncharged and assist in peptide binding. In L3MBTL1, the corresponding residues (Met357, Asp383 and Asp415) can also assist in peptide binding, however, the negatively charged area around the methyl-lysine-binding pocket is less extended compared with dSfmbt (Figure 3B), which might explain the lower binding affinity. Multiple binding sites in MBT-repeat proteins Superposition of the fourth MBT repeat of dSfmbt with the three other repeats (Figure 4) shows that only the fourth repeat can accommodate methyl-lysine residues. In repeat 1, the crucial aspartate is substituted by an asparagine (Figure 1C), but more importantly the conformation of the loop bearing this residue is different. In the second repeat, two of the cage-forming aromatic residues are substituted by aspartate and serine, respectively, and in the third repeat, Tyr836 blocks the access of the methyl-lysine to the binding pocket. The MBT proteins, Scm and L3MBTL1, use their second MBT repeat for methyl-lysine binding and, indeed, the cage-forming residues, including Cys925 are well conserved in the second MBT repeat of L3MBTL1 and in the second repeat of Scm. In contrast, in MBT repeats 1 and 3 of human L3MBTL1, Cys925 is substituted by bulkier residues that block the access to the binding pocket, whereas in Scm the cage-forming aromatic residues are substituted by smaller residues. In dSfmbt and Scm, conserved residues cluster around the methyl-lysine binding pocket, whereas the patch of strictly conserved residues is smaller in L3MBTL1 (Figure 3C). Figure 4.Stereo view of superpositons of the MBT-repeat domains of dSfmbt, L3MBTL1 and Scm. Colour code corresponds to Figure 3 with dSfmbt repeats 1, 2, 3 and 4 depicted in blue, green, yellow, and red (top), L3MBTL1 repeats 1, 2 and 3 in green, yellow and red (middle) and Scm repeats 1 and 2 depicted in green and red (bottom). Download figure Download PowerPoint In conclusion, only a single MBT repeat in Scm, L3MBTL1 and dSfmbt can bind mono- and dimethylated lysine residues. It is possible that the other MBT repeats recognize other ligands. Indeed, in one of the crystal structures of L3MBTL1, the first MBT repeat binds a Pro–Ser-motif-containing peptide of a neighbouring molecule (Li et al, 2007), although the functional relevance of this interaction is not known. dSfmbt and Scm interact functionally to maintain Polycomb repression Previous structural/functional analyses of the MBT-repeat domain of Scm showed that a point mutation in the methyl-lysine-binding pocket that abolishes the methyl-lysine binding, or even complete deletion of the MBT-repeat domain, still permit these mutant Scm proteins to partially maintain PcG repression of target genes in a genetic-rescue assay in D. melanogaster (Grimm et al, 2007). Similar observations were made with dSfmbt; we found that not only the wild-type dSfmbt protein but also the dSfmbtE947A/Y948F/D917A protein (see above) is able to maintain PcG repression of target genes in a genetic-rescue assay in dSfmbt null mutants (data not shown). One possible explanation for these findings would be that methyl-lysine binding by the MBT domains of dSfmbt and Scm has only a minor function in PcG repression. However, as both the proteins have similar methyl-lysine-binding activities, an alternative possibility could be that the MBT-repeat domains in Scm and dSfmbt function in a partially redundant manner to maintain PcG repression. We there
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