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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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Cofactor binding pocket. (A) A representative simulated-annealing omit Fo–Fc electron density map (contoured at 2.0 σ level) for the cofactor product AdoHcy. (B) A detailed comparison of the cofactor binding mode of Smyd3 (left panel) with that of SET7/9 (right panel, PDB code 1O9S). The hydrogen bonds are indicated with black dotted lines. The residues contributing to the cofactor binding with their side chains are labeled in orange, and those with their backbones in black. Depending on the contributing moieties, the side chains or backbones of the residues involved in the AdoHcy binding are shown with ball-and-stick models. The color coding is the same as in Figure 1A. (C) HKMT activity assays of the wild-type Smyd3 and the mutants carrying point mutations at the cofactor binding pocket.
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Figure 3: Cofactor binding pocket. (A) A representative simulated-annealing omit Fo–Fc electron density map (contoured at 2.0 σ level) for the cofactor product AdoHcy. (B) A detailed comparison of the cofactor binding mode of Smyd3 (left panel) with that of SET7/9 (right panel, PDB code 1O9S). The hydrogen bonds are indicated with black dotted lines. The residues contributing to the cofactor binding with their side chains are labeled in orange, and those with their backbones in black. Depending on the contributing moieties, the side chains or backbones of the residues involved in the AdoHcy binding are shown with ball-and-stick models. The color coding is the same as in Figure 1A. (C) HKMT activity assays of the wild-type Smyd3 and the mutants carrying point mutations at the cofactor binding pocket.

Mentions: At the active site of the Smyd3–AdoHcy structure, AdoHcy is bound in a pocket surrounded by the β1–β2 loop, the η1–η2 loop and β9 of the SET domain and the α6–α7 loop of the post-SET domain (Figure 1B) with well-defined electron density (Figure 3A). Similar to AdoHcy bound in the SET7/9 structures (18,25), the cofactor takes a U-shape conformation in the Smyd3 structure (Figure 3B). The adenine ring of AdoHcy makes a π–π stacking interaction with the side chain of Phe259, and the N6 atom of the adenine forms a hydrogen bond with the main-chain carbonyl of His206. For stabilization of the ribose ring, the O2′ atom forms a hydrogen bond with the side-chain amino group of Asn132, and the O3′ atom forms hydrogen bonds with the side-chain carbonyl of Asn132 and the main-chain carbonyl of Tyr257. The amide group of the homocysteine is hydrogen-bonded to the main-chain carbonyl groups of Arg14 and Asn16 and the side-chain carbonyl of Asn205. The carboxyl group forms hydrogen bonds with the main-chain amide of Asn16 and the phenolic hydroxyl of Tyr124.Figure 3.


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)

Cofactor binding pocket. (A) A representative simulated-annealing omit Fo–Fc electron density map (contoured at 2.0 σ level) for the cofactor product AdoHcy. (B) A detailed comparison of the cofactor binding mode of Smyd3 (left panel) with that of SET7/9 (right panel, PDB code 1O9S). The hydrogen bonds are indicated with black dotted lines. The residues contributing to the cofactor binding with their side chains are labeled in orange, and those with their backbones in black. Depending on the contributing moieties, the side chains or backbones of the residues involved in the AdoHcy binding are shown with ball-and-stick models. The color coding is the same as in Figure 1A. (C) HKMT activity assays of the wild-type Smyd3 and the mutants carrying point mutations at the cofactor binding pocket.
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Related In: Results  -  Collection

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Figure 3: Cofactor binding pocket. (A) A representative simulated-annealing omit Fo–Fc electron density map (contoured at 2.0 σ level) for the cofactor product AdoHcy. (B) A detailed comparison of the cofactor binding mode of Smyd3 (left panel) with that of SET7/9 (right panel, PDB code 1O9S). The hydrogen bonds are indicated with black dotted lines. The residues contributing to the cofactor binding with their side chains are labeled in orange, and those with their backbones in black. Depending on the contributing moieties, the side chains or backbones of the residues involved in the AdoHcy binding are shown with ball-and-stick models. The color coding is the same as in Figure 1A. (C) HKMT activity assays of the wild-type Smyd3 and the mutants carrying point mutations at the cofactor binding pocket.
Mentions: At the active site of the Smyd3–AdoHcy structure, AdoHcy is bound in a pocket surrounded by the β1–β2 loop, the η1–η2 loop and β9 of the SET domain and the α6–α7 loop of the post-SET domain (Figure 1B) with well-defined electron density (Figure 3A). Similar to AdoHcy bound in the SET7/9 structures (18,25), the cofactor takes a U-shape conformation in the Smyd3 structure (Figure 3B). The adenine ring of AdoHcy makes a π–π stacking interaction with the side chain of Phe259, and the N6 atom of the adenine forms a hydrogen bond with the main-chain carbonyl of His206. For stabilization of the ribose ring, the O2′ atom forms a hydrogen bond with the side-chain amino group of Asn132, and the O3′ atom forms hydrogen bonds with the side-chain carbonyl of Asn132 and the main-chain carbonyl of Tyr257. The amide group of the homocysteine is hydrogen-bonded to the main-chain carbonyl groups of Arg14 and Asn16 and the side-chain carbonyl of Asn205. The carboxyl group forms hydrogen bonds with the main-chain amide of Asn16 and the phenolic hydroxyl of Tyr124.Figure 3.

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