Mechanism of histone lysine methyl transfer revealed by the structure of SET7/9-AdoMet

Abstract

The methylation of lysine residues of histones plays a pivotal role in the regulation of chromatin structure and gene expression. Here, we report two crystal structures of SET7/9, a histone methyltransferase (HMTase) that transfers methyl groups to Lys4 of histone H3, in complex with S-adenosyl-L-methionine (AdoMet) determined at 1.7 and 2.3 A resolution. The structures reveal an active site consisting of: (i) a binding pocket between the SET domain and a c-SET helix where an AdoMet molecule in an unusual conformation binds; (ii) a narrow substrate-specific channel that only unmethylated lysine residues can access; and (iii) a catalytic tyrosine residue. The methyl group of AdoMet is directed to the narrow channel where a substrate lysine enters from the opposite side. We demonstrate that SET7/9 can transfer two but not three methyl groups to unmodified Lys4 of H3 without substrate dissociation. The unusual features of the SET domain-containing HMTase discriminate between the un- and methylated lysine substrate, and the methylation sites for the histone H3 tail.

Fig. 1. (A) Stereo diagram of the 1.7 Å resolution electron density map around the methyl group of AdoMet. The |F_o – F_c| simulated annealing omit map contoured at the 3σ level, with AdoMet and neighbouring residues omitted for map calculation. The refined 1.7 Å structure is superimposed on the map. (B) Sequence alignment of SET7/9, human SUV39H1 and the yeast homologue Clr4. Residues identical between SET7/9, SUV39H1 and Clr4 are highlighted in yellow. The bar graph below the sequence alignment indicates the degree of identity among 11 SET domain-containing HMTases: Hs SET9, Mm Suv39h1, Hs Suv39H1, Dm Su(var)3–9, Hs G9a, Sc SET1, Sp Clr4, Nc dim-5, Hs PR-SET7, Sc SET2, Mm ESET and Hs ESET. Insertions in SUV39H1 and Clr4 sequences are dropped below the alignment with a vertical line for clarity. Loops that form the active site pocket are highlighted in orange, and residues that make up the pocket walls and active site, and those that contact AdoMet are indicated.

Fig. 1. (A) Stereo diagram of the 1.7 Å resolution electron density map around the methyl group of AdoMet. The |F_o – F_c| simulated annealing omit map contoured at the 3σ level, with AdoMet and neighbouring residues omitted for map calculation. The refined 1.7 Å structure is superimposed on the map. (B) Sequence alignment of SET7/9, human SUV39H1 and the yeast homologue Clr4. Residues identical between SET7/9, SUV39H1 and Clr4 are highlighted in yellow. The bar graph below the sequence alignment indicates the degree of identity among 11 SET domain-containing HMTases: Hs SET9, Mm Suv39h1, Hs Suv39H1, Dm Su(var)3–9, Hs G9a, Sc SET1, Sp Clr4, Nc dim-5, Hs PR-SET7, Sc SET2, Mm ESET and Hs ESET. Insertions in SUV39H1 and Clr4 sequences are dropped below the alignment with a vertical line for clarity. Loops that form the active site pocket are highlighted in orange, and residues that make up the pocket walls and active site, and those that contact AdoMet are indicated.

Fig. 2. Overall structure of SET7/9–AdoMet. (A) The 2.3 Å SET7/9L structure is shown in a ribbon representation. A bound AdoMet molecule is shown in a ball-and-stick model (oxygen, red; carbon, cyan; nitrogen, blue; and sulfur, green). The n-, SET and c-SET regions in the C-terminal domain are coloured in yellow, red and green, respectively. (B) Topological diagrams of secondary structure elements. Loops forming the active site pocket are in red. The AdoMet-binding site is indicated. The colour scheme is identical to that in (A). (C) Stereo diagram showing an extensive van der Waals contacts network between the SET and c-SET region. The AdoMet is coloured in cyan, and the residues from SET and c-SET domains are in yellow. Other atoms are shown in the same colour as in (A).

Fig. 2. Overall structure of SET7/9–AdoMet. (A) The 2.3 Å SET7/9L structure is shown in a ribbon representation. A bound AdoMet molecule is shown in a ball-and-stick model (oxygen, red; carbon, cyan; nitrogen, blue; and sulfur, green). The n-, SET and c-SET regions in the C-terminal domain are coloured in yellow, red and green, respectively. (B) Topological diagrams of secondary structure elements. Loops forming the active site pocket are in red. The AdoMet-binding site is indicated. The colour scheme is identical to that in (A). (C) Stereo diagram showing an extensive van der Waals contacts network between the SET and c-SET region. The AdoMet is coloured in cyan, and the residues from SET and c-SET domains are in yellow. Other atoms are shown in the same colour as in (A).

Fig. 2. Overall structure of SET7/9–AdoMet. (A) The 2.3 Å SET7/9L structure is shown in a ribbon representation. A bound AdoMet molecule is shown in a ball-and-stick model (oxygen, red; carbon, cyan; nitrogen, blue; and sulfur, green). The n-, SET and c-SET regions in the C-terminal domain are coloured in yellow, red and green, respectively. (B) Topological diagrams of secondary structure elements. Loops forming the active site pocket are in red. The AdoMet-binding site is indicated. The colour scheme is identical to that in (A). (C) Stereo diagram showing an extensive van der Waals contacts network between the SET and c-SET region. The AdoMet is coloured in cyan, and the residues from SET and c-SET domains are in yellow. Other atoms are shown in the same colour as in (A).

Fig. 3. The AdoMet-binding pocket. (A) Surface representation of the AdoMet-binding pocket. The surface is coloured according to the residue identity as in Figure 1B, highlighting the conservation of residues that make up the AdoMet-binding site. The AdoMet molecule is shown in a bond representation. The orientation of the figure is similar to that in (B). (B) The AdoMet-binding pocket in SET7/9S. Key residues discussed in the text are shown in a ball-and-stick model. The AdoMet molecule is shown with same colour scheme as in (A), except that carbon atoms are shown in cyan. (C) Stereo diagram showing the conformations of AdoMet molecules. Three AdoMet molecules, extended (red), intermediate (blue) and compact (yellow) forms, are superimposed. The extended AdoMet molecule was drawn using AdoMet bound to isoflavone-O-methyltransferase [Protein data bank (PDB) identifiaction code: 1FPX], and the intermediate form was from AdoMet bound to methionine repressor protein (Metj; PDB identification code: 1CMA). (D) A schematic drawing showing direct interactions between SET7/9 and AdoMet. Hydrogen bonds are shown by dashed lines. An arc next to the residue name indicates that the amino acid is involved in a van der Waals interaction with AdoMet. The residues coloured in blue indicate side chain interactions, while those in red are involved in backbone interactions.

Fig. 3. The AdoMet-binding pocket. (A) Surface representation of the AdoMet-binding pocket. The surface is coloured according to the residue identity as in Figure 1B, highlighting the conservation of residues that make up the AdoMet-binding site. The AdoMet molecule is shown in a bond representation. The orientation of the figure is similar to that in (B). (B) The AdoMet-binding pocket in SET7/9S. Key residues discussed in the text are shown in a ball-and-stick model. The AdoMet molecule is shown with same colour scheme as in (A), except that carbon atoms are shown in cyan. (C) Stereo diagram showing the conformations of AdoMet molecules. Three AdoMet molecules, extended (red), intermediate (blue) and compact (yellow) forms, are superimposed. The extended AdoMet molecule was drawn using AdoMet bound to isoflavone-O-methyltransferase [Protein data bank (PDB) identifiaction code: 1FPX], and the intermediate form was from AdoMet bound to methionine repressor protein (Metj; PDB identification code: 1CMA). (D) A schematic drawing showing direct interactions between SET7/9 and AdoMet. Hydrogen bonds are shown by dashed lines. An arc next to the residue name indicates that the amino acid is involved in a van der Waals interaction with AdoMet. The residues coloured in blue indicate side chain interactions, while those in red are involved in backbone interactions.

Fig. 3. The AdoMet-binding pocket. (A) Surface representation of the AdoMet-binding pocket. The surface is coloured according to the residue identity as in Figure 1B, highlighting the conservation of residues that make up the AdoMet-binding site. The AdoMet molecule is shown in a bond representation. The orientation of the figure is similar to that in (B). (B) The AdoMet-binding pocket in SET7/9S. Key residues discussed in the text are shown in a ball-and-stick model. The AdoMet molecule is shown with same colour scheme as in (A), except that carbon atoms are shown in cyan. (C) Stereo diagram showing the conformations of AdoMet molecules. Three AdoMet molecules, extended (red), intermediate (blue) and compact (yellow) forms, are superimposed. The extended AdoMet molecule was drawn using AdoMet bound to isoflavone-O-methyltransferase [Protein data bank (PDB) identifiaction code: 1FPX], and the intermediate form was from AdoMet bound to methionine repressor protein (Metj; PDB identification code: 1CMA). (D) A schematic drawing showing direct interactions between SET7/9 and AdoMet. Hydrogen bonds are shown by dashed lines. An arc next to the residue name indicates that the amino acid is involved in a van der Waals interaction with AdoMet. The residues coloured in blue indicate side chain interactions, while those in red are involved in backbone interactions.

Fig. 3. The AdoMet-binding pocket. (A) Surface representation of the AdoMet-binding pocket. The surface is coloured according to the residue identity as in Figure 1B, highlighting the conservation of residues that make up the AdoMet-binding site. The AdoMet molecule is shown in a bond representation. The orientation of the figure is similar to that in (B). (B) The AdoMet-binding pocket in SET7/9S. Key residues discussed in the text are shown in a ball-and-stick model. The AdoMet molecule is shown with same colour scheme as in (A), except that carbon atoms are shown in cyan. (C) Stereo diagram showing the conformations of AdoMet molecules. Three AdoMet molecules, extended (red), intermediate (blue) and compact (yellow) forms, are superimposed. The extended AdoMet molecule was drawn using AdoMet bound to isoflavone-O-methyltransferase [Protein data bank (PDB) identifiaction code: 1FPX], and the intermediate form was from AdoMet bound to methionine repressor protein (Metj; PDB identification code: 1CMA). (D) A schematic drawing showing direct interactions between SET7/9 and AdoMet. Hydrogen bonds are shown by dashed lines. An arc next to the residue name indicates that the amino acid is involved in a van der Waals interaction with AdoMet. The residues coloured in blue indicate side chain interactions, while those in red are involved in backbone interactions.

Fig. 4. A channel at the active site pocket. (A) A surface representation showing the location of the putative lysine-binding channel and a conserved shallow groove for the substrate-binding site (indicated by an arrow). See the text for sites 1, 2 and 3. The colouring scheme is identical to that in Figure 3A. AdoMet is shown in a bond model. The N-terminal domain is not coloured since it is not homologous to those of other SET-containing HMTases (Figure 1B). (B) A diagram showing the substrate-specific channel in SET7/9S. Key residues discussed in the text are shown in a ball-and-stick model. The AdoMet molecule is shown with the same colour scheme as in Figure 3B. (C) SET7/9 methylates unmethylated H3 peptide but cannot add methyl group(s) to already methylated peptide. An N-terminal peptide with amino acids 1–8 of unmodified, mono- or dimethylated H3-K4 was used for the assay of SET7/9 HMTase activity. The full-length SET7/9 was used for the assay. (D) Methylation specificity of SET7/9. Histone H3 (Roche) was methylated by SET7/9. The reaction products were resolved by SDS–PAGE, blotted to nitrocellulose and probed with either a H3-K4 mono- or dimethyl antibody, or a H3-K4 trimethyl antibody as indicated. The H3-K4, K9 trimethyl antibody also gave the same result as that of the H3-K4 trimethyl antibody (data not shown).

Fig. 4. A channel at the active site pocket. (A) A surface representation showing the location of the putative lysine-binding channel and a conserved shallow groove for the substrate-binding site (indicated by an arrow). See the text for sites 1, 2 and 3. The colouring scheme is identical to that in Figure 3A. AdoMet is shown in a bond model. The N-terminal domain is not coloured since it is not homologous to those of other SET-containing HMTases (Figure 1B). (B) A diagram showing the substrate-specific channel in SET7/9S. Key residues discussed in the text are shown in a ball-and-stick model. The AdoMet molecule is shown with the same colour scheme as in Figure 3B. (C) SET7/9 methylates unmethylated H3 peptide but cannot add methyl group(s) to already methylated peptide. An N-terminal peptide with amino acids 1–8 of unmodified, mono- or dimethylated H3-K4 was used for the assay of SET7/9 HMTase activity. The full-length SET7/9 was used for the assay. (D) Methylation specificity of SET7/9. Histone H3 (Roche) was methylated by SET7/9. The reaction products were resolved by SDS–PAGE, blotted to nitrocellulose and probed with either a H3-K4 mono- or dimethyl antibody, or a H3-K4 trimethyl antibody as indicated. The H3-K4, K9 trimethyl antibody also gave the same result as that of the H3-K4 trimethyl antibody (data not shown).

Fig. 4. A channel at the active site pocket. (A) A surface representation showing the location of the putative lysine-binding channel and a conserved shallow groove for the substrate-binding site (indicated by an arrow). See the text for sites 1, 2 and 3. The colouring scheme is identical to that in Figure 3A. AdoMet is shown in a bond model. The N-terminal domain is not coloured since it is not homologous to those of other SET-containing HMTases (Figure 1B). (B) A diagram showing the substrate-specific channel in SET7/9S. Key residues discussed in the text are shown in a ball-and-stick model. The AdoMet molecule is shown with the same colour scheme as in Figure 3B. (C) SET7/9 methylates unmethylated H3 peptide but cannot add methyl group(s) to already methylated peptide. An N-terminal peptide with amino acids 1–8 of unmodified, mono- or dimethylated H3-K4 was used for the assay of SET7/9 HMTase activity. The full-length SET7/9 was used for the assay. (D) Methylation specificity of SET7/9. Histone H3 (Roche) was methylated by SET7/9. The reaction products were resolved by SDS–PAGE, blotted to nitrocellulose and probed with either a H3-K4 mono- or dimethyl antibody, or a H3-K4 trimethyl antibody as indicated. The H3-K4, K9 trimethyl antibody also gave the same result as that of the H3-K4 trimethyl antibody (data not shown).

Fig. 5. (A) Stereo diagram showing the active site region in SET7/9S. Eight residues discussed in the text along with interacting residues are shown in a ball-and-stick model. (B) HMTase activities of the SET7/9 point mutants. Reactions contained wild-type or mutant SET7/9, 1 µM AdoMet and 1 µg of H3 from calf thymus. Assays were performed in triplicate.

Fig. 5. (A) Stereo diagram showing the active site region in SET7/9S. Eight residues discussed in the text along with interacting residues are shown in a ball-and-stick model. (B) HMTase activities of the SET7/9 point mutants. Reactions contained wild-type or mutant SET7/9, 1 µM AdoMet and 1 µg of H3 from calf thymus. Assays were performed in triplicate.