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Structural and biochemical studies of human lysine methyltransferase Smyd3 reveal the important functional roles of its post-SET and TPR domains and the regulation of its activity by DNA binding.

Xu S, Wu J, Sun B, Zhong C, Ding J - Nucleic Acids Res. (2011)

Bottom Line: Our data demonstrate the important roles of both TPR and post-SET domains in the histone lysine methyltransferase (HKMT) activity of Smyd3, and show that the hydroxyl group of Tyr239 is critical for the enzymatic activity.The characteristic MYND domain is located nearby to the substrate binding pocket and exhibits a largely positively charged surface.Further biochemical assays show that DNA binding of Smyd3 can stimulate its HKMT activity and the process may be mediated via the MYND domain through direct DNA binding.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Biology and Research Center for Structural Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.

ABSTRACT
The SET- and MYND-domain containing (Smyd) proteins constitute a special subfamily of the SET-containing lysine methyltransferases. Here we present the structure of full-length human Smyd3 in complex with S-adenosyl-L-homocysteine at 2.8 Å resolution. Smyd3 affords the first example that other region(s) besides the SET domain and its flanking regions participate in the formation of the active site. Structural analysis shows that the previously uncharacterized C-terminal domain of Smyd3 contains a tetratrico-peptide repeat (TPR) domain which together with the SET and post-SET domains forms a deep, narrow substrate binding pocket. Our data demonstrate the important roles of both TPR and post-SET domains in the histone lysine methyltransferase (HKMT) activity of Smyd3, and show that the hydroxyl group of Tyr239 is critical for the enzymatic activity. The characteristic MYND domain is located nearby to the substrate binding pocket and exhibits a largely positively charged surface. Further biochemical assays show that DNA binding of Smyd3 can stimulate its HKMT activity and the process may be mediated via the MYND domain through direct DNA binding.

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Related in: MedlinePlus

Structure of the Smyd3–AdoHcy complex. (A) Overall structure of the Smyd3–AdoHcy complex. Top: a schematic representation of the full-length Smyd3 with the N-terminal SET domain (residues 1–43 and 94–242), the MYND domain (residues 44–93), the post-SET domain (residues 243–270), and the C-terminal region (residues 271–428) colored in magenta, yellow, cyan and blue, respectively. Bottom: two views of the overall structure of the Smyd3–AdoHcy complex. The domains are colored accordingly and the secondary structure elements are marked. The cofactor product AdoHcy is shown with a ball-and-stick model and colored in cyan. (B) Structural comparison of the SET and post-SET domains of Smyd3 with the equivalent regions of SET7/9 (PDB code 1O9S). Superposition of the Smyd3 and Set7/9 structures was performed based on the core region of the SET domain. SET7/9 is colored in green, and the color coding for Smyd3 is the same as in Figure 1A. The cofactors are shown with ball-and-stick models and colored accordingly. (C) Comparison of the Zn2+-binding site in the catalytic core of Smyd3 with the equivalent regions of SET7/9 (left panel) and Dim 5 (PDB code 1PEG, right panel). The post-SET regions of Smyd3, SET7/9 and Dim5 are shown with ribbon representations and colored in cyan, green and wheat, respectively. The side chains of the involved Cys residues and the bound cofactors are shown with ball-and-stick models and colored accordingly. The Zn2+ ions are shown with sphere models and colored accordingly. The secondary structure elements and the involved Cys residues in Smyd3 are labeled. (D) Zinc-binding sites in the MYND domain. The MYND domain (yellow) is characterized by a C6HC zinc chelating motif. The side chains of the Cys and His residues chelating the two Zn2+ ions are shown with ball-and-stick models. The Zn2+ ions are shown with sphere models. The secondary structure elements and the involved residues are labeled.
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Figure 1: Structure of the Smyd3–AdoHcy complex. (A) Overall structure of the Smyd3–AdoHcy complex. Top: a schematic representation of the full-length Smyd3 with the N-terminal SET domain (residues 1–43 and 94–242), the MYND domain (residues 44–93), the post-SET domain (residues 243–270), and the C-terminal region (residues 271–428) colored in magenta, yellow, cyan and blue, respectively. Bottom: two views of the overall structure of the Smyd3–AdoHcy complex. The domains are colored accordingly and the secondary structure elements are marked. The cofactor product AdoHcy is shown with a ball-and-stick model and colored in cyan. (B) Structural comparison of the SET and post-SET domains of Smyd3 with the equivalent regions of SET7/9 (PDB code 1O9S). Superposition of the Smyd3 and Set7/9 structures was performed based on the core region of the SET domain. SET7/9 is colored in green, and the color coding for Smyd3 is the same as in Figure 1A. The cofactors are shown with ball-and-stick models and colored accordingly. (C) Comparison of the Zn2+-binding site in the catalytic core of Smyd3 with the equivalent regions of SET7/9 (left panel) and Dim 5 (PDB code 1PEG, right panel). The post-SET regions of Smyd3, SET7/9 and Dim5 are shown with ribbon representations and colored in cyan, green and wheat, respectively. The side chains of the involved Cys residues and the bound cofactors are shown with ball-and-stick models and colored accordingly. The Zn2+ ions are shown with sphere models and colored accordingly. The secondary structure elements and the involved Cys residues in Smyd3 are labeled. (D) Zinc-binding sites in the MYND domain. The MYND domain (yellow) is characterized by a C6HC zinc chelating motif. The side chains of the Cys and His residues chelating the two Zn2+ ions are shown with ball-and-stick models. The Zn2+ ions are shown with sphere models. The secondary structure elements and the involved residues are labeled.

Mentions: Crystallization of the full-length human Smyd3 in complex with the cofactor product AdoHcy was carried out, and three different forms of crystals belonging to three different space groups (P21, P212121 and P61) have been obtained (Table 1). The structures derived from the three forms of crystals are all similar with AdoHcy and three Zn2+ ions bound at similar positions, and the one (form I) refined to the highest resolution (2.8 Å) was used for further structural analysis and discussion. As shown in Figure 1A and B, the Smyd3–AdoHcy complex assumes a compact globular structure. The N-terminal region including the SET domain (residues 1–43 and 94–242), the MYND domain (residues 44–93) and the post-SET domain (residues 243–270) has a mixed structure consisting of α helices (α1–α7), β-strands (β1–β12) and long extended loops, while the C-terminal region (residues 271–428) is comprised of mainly α helices (α8–α15) (Figure 1A and Supplementary Figure S2). Our Smyd3–AdoHcy structure is very similar to the Smyd3 structure in complex with AdoMet (PDB code 3MEK) which was derived from a crystal of space group P212121 with an RMSD of 0.9 Å for all Cα atoms. In addition, the structures of the active site are almost identical in the two complexes with AdoHcy and AdoMet bound in a similar mode.Figure 1.


Structural and biochemical studies of human lysine methyltransferase Smyd3 reveal the important functional roles of its post-SET and TPR domains and the regulation of its activity by DNA binding.

Xu S, Wu J, Sun B, Zhong C, Ding J - Nucleic Acids Res. (2011)

Structure of the Smyd3–AdoHcy complex. (A) Overall structure of the Smyd3–AdoHcy complex. Top: a schematic representation of the full-length Smyd3 with the N-terminal SET domain (residues 1–43 and 94–242), the MYND domain (residues 44–93), the post-SET domain (residues 243–270), and the C-terminal region (residues 271–428) colored in magenta, yellow, cyan and blue, respectively. Bottom: two views of the overall structure of the Smyd3–AdoHcy complex. The domains are colored accordingly and the secondary structure elements are marked. The cofactor product AdoHcy is shown with a ball-and-stick model and colored in cyan. (B) Structural comparison of the SET and post-SET domains of Smyd3 with the equivalent regions of SET7/9 (PDB code 1O9S). Superposition of the Smyd3 and Set7/9 structures was performed based on the core region of the SET domain. SET7/9 is colored in green, and the color coding for Smyd3 is the same as in Figure 1A. The cofactors are shown with ball-and-stick models and colored accordingly. (C) Comparison of the Zn2+-binding site in the catalytic core of Smyd3 with the equivalent regions of SET7/9 (left panel) and Dim 5 (PDB code 1PEG, right panel). The post-SET regions of Smyd3, SET7/9 and Dim5 are shown with ribbon representations and colored in cyan, green and wheat, respectively. The side chains of the involved Cys residues and the bound cofactors are shown with ball-and-stick models and colored accordingly. The Zn2+ ions are shown with sphere models and colored accordingly. The secondary structure elements and the involved Cys residues in Smyd3 are labeled. (D) Zinc-binding sites in the MYND domain. The MYND domain (yellow) is characterized by a C6HC zinc chelating motif. The side chains of the Cys and His residues chelating the two Zn2+ ions are shown with ball-and-stick models. The Zn2+ ions are shown with sphere models. The secondary structure elements and the involved residues are labeled.
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: Structure of the Smyd3–AdoHcy complex. (A) Overall structure of the Smyd3–AdoHcy complex. Top: a schematic representation of the full-length Smyd3 with the N-terminal SET domain (residues 1–43 and 94–242), the MYND domain (residues 44–93), the post-SET domain (residues 243–270), and the C-terminal region (residues 271–428) colored in magenta, yellow, cyan and blue, respectively. Bottom: two views of the overall structure of the Smyd3–AdoHcy complex. The domains are colored accordingly and the secondary structure elements are marked. The cofactor product AdoHcy is shown with a ball-and-stick model and colored in cyan. (B) Structural comparison of the SET and post-SET domains of Smyd3 with the equivalent regions of SET7/9 (PDB code 1O9S). Superposition of the Smyd3 and Set7/9 structures was performed based on the core region of the SET domain. SET7/9 is colored in green, and the color coding for Smyd3 is the same as in Figure 1A. The cofactors are shown with ball-and-stick models and colored accordingly. (C) Comparison of the Zn2+-binding site in the catalytic core of Smyd3 with the equivalent regions of SET7/9 (left panel) and Dim 5 (PDB code 1PEG, right panel). The post-SET regions of Smyd3, SET7/9 and Dim5 are shown with ribbon representations and colored in cyan, green and wheat, respectively. The side chains of the involved Cys residues and the bound cofactors are shown with ball-and-stick models and colored accordingly. The Zn2+ ions are shown with sphere models and colored accordingly. The secondary structure elements and the involved Cys residues in Smyd3 are labeled. (D) Zinc-binding sites in the MYND domain. The MYND domain (yellow) is characterized by a C6HC zinc chelating motif. The side chains of the Cys and His residues chelating the two Zn2+ ions are shown with ball-and-stick models. The Zn2+ ions are shown with sphere models. The secondary structure elements and the involved residues are labeled.
Mentions: Crystallization of the full-length human Smyd3 in complex with the cofactor product AdoHcy was carried out, and three different forms of crystals belonging to three different space groups (P21, P212121 and P61) have been obtained (Table 1). The structures derived from the three forms of crystals are all similar with AdoHcy and three Zn2+ ions bound at similar positions, and the one (form I) refined to the highest resolution (2.8 Å) was used for further structural analysis and discussion. As shown in Figure 1A and B, the Smyd3–AdoHcy complex assumes a compact globular structure. The N-terminal region including the SET domain (residues 1–43 and 94–242), the MYND domain (residues 44–93) and the post-SET domain (residues 243–270) has a mixed structure consisting of α helices (α1–α7), β-strands (β1–β12) and long extended loops, while the C-terminal region (residues 271–428) is comprised of mainly α helices (α8–α15) (Figure 1A and Supplementary Figure S2). Our Smyd3–AdoHcy structure is very similar to the Smyd3 structure in complex with AdoMet (PDB code 3MEK) which was derived from a crystal of space group P212121 with an RMSD of 0.9 Å for all Cα atoms. In addition, the structures of the active site are almost identical in the two complexes with AdoHcy and AdoMet bound in a similar mode.Figure 1.

Bottom Line: Our data demonstrate the important roles of both TPR and post-SET domains in the histone lysine methyltransferase (HKMT) activity of Smyd3, and show that the hydroxyl group of Tyr239 is critical for the enzymatic activity.The characteristic MYND domain is located nearby to the substrate binding pocket and exhibits a largely positively charged surface.Further biochemical assays show that DNA binding of Smyd3 can stimulate its HKMT activity and the process may be mediated via the MYND domain through direct DNA binding.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Biology and Research Center for Structural Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.

ABSTRACT
The SET- and MYND-domain containing (Smyd) proteins constitute a special subfamily of the SET-containing lysine methyltransferases. Here we present the structure of full-length human Smyd3 in complex with S-adenosyl-L-homocysteine at 2.8 Å resolution. Smyd3 affords the first example that other region(s) besides the SET domain and its flanking regions participate in the formation of the active site. Structural analysis shows that the previously uncharacterized C-terminal domain of Smyd3 contains a tetratrico-peptide repeat (TPR) domain which together with the SET and post-SET domains forms a deep, narrow substrate binding pocket. Our data demonstrate the important roles of both TPR and post-SET domains in the histone lysine methyltransferase (HKMT) activity of Smyd3, and show that the hydroxyl group of Tyr239 is critical for the enzymatic activity. The characteristic MYND domain is located nearby to the substrate binding pocket and exhibits a largely positively charged surface. Further biochemical assays show that DNA binding of Smyd3 can stimulate its HKMT activity and the process may be mediated via the MYND domain through direct DNA binding.

Show MeSH
Related in: MedlinePlus