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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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Histone binding pocket. (A) Potential histone peptide binding site. Superposition of the Smyd3 and SET7/9 (PDB code 1O9S) structures was performed as in Figure 1B. Smyd3 is shown in a ribbon representation with the same color coding as in Figure 1A. For simplicity, for SET7/9 only the bound histone peptide is shown in a ribbon representation and colored in green and the side chain of the methyllysine of the histone peptide is shown with a ball-and-stick model and colored accordingly. The cofactor is also shown with a ball-and-stick model. (B) Electrostatic potential surface of the potential histone peptide binding pocket in Smyd3. The surface charge distribution is displayed as blue for positive, red for negative, and white for neutral. A close-up view of the pocket (middle panel) shows that several acidic patches are present at the opening of the binding pocket. Some of the acidic residues in these patches are labeled. The TPR domain forms part of the substrate binding pocket and removal of the TPR domain would leave an incomplete pocket (right panel). (C) HKMT activity assays of the wild-type Smyd3 and the mutants with truncation or mutations at the substrate binding pocket. The activities of the truncate with deletion of the C-terminal region (Δ277–428) and the mutants carrying one or two point mutations of the residues potentially involved in histone binding were determined. Activity is shown as the relative activity of the proteins normalized to that of the wild-type protein. The experiments were performed in triplicates and the error bars indicate the standard deviation.
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Figure 2: Histone binding pocket. (A) Potential histone peptide binding site. Superposition of the Smyd3 and SET7/9 (PDB code 1O9S) structures was performed as in Figure 1B. Smyd3 is shown in a ribbon representation with the same color coding as in Figure 1A. For simplicity, for SET7/9 only the bound histone peptide is shown in a ribbon representation and colored in green and the side chain of the methyllysine of the histone peptide is shown with a ball-and-stick model and colored accordingly. The cofactor is also shown with a ball-and-stick model. (B) Electrostatic potential surface of the potential histone peptide binding pocket in Smyd3. The surface charge distribution is displayed as blue for positive, red for negative, and white for neutral. A close-up view of the pocket (middle panel) shows that several acidic patches are present at the opening of the binding pocket. Some of the acidic residues in these patches are labeled. The TPR domain forms part of the substrate binding pocket and removal of the TPR domain would leave an incomplete pocket (right panel). (C) HKMT activity assays of the wild-type Smyd3 and the mutants with truncation or mutations at the substrate binding pocket. The activities of the truncate with deletion of the C-terminal region (Δ277–428) and the mutants carrying one or two point mutations of the residues potentially involved in histone binding were determined. Activity is shown as the relative activity of the proteins normalized to that of the wild-type protein. The experiments were performed in triplicates and the error bars indicate the standard deviation.

Mentions: Our attempts to obtain a structure of Smyd3 in complex with the histone peptide were unsuccessful. The Smyd3–AdoHcy structure was superposed to the structure of SET7/9 in complex with a methylated histone peptide (PDB code 1O9S) (25) (Figures 1B and 2A). In SET7/9, a narrow lysine channel is detected connecting the cofactor binding site and the histone peptide binding site (25). In the Smyd3–AdoHcy complex, a similar channel is present (see details later) and at one end of the channel AdoHcy binds at an almost identical position as in the SET7/9 complex (Figure 1B). Thus, we reason that the histone substrate of Smyd3 should bind at the other end of the channel as observed in SET7/9. In the SET7/9 structures, the histone peptide binding site is quite open as the N-terminal pre-SET domain is distant from the active site (21,25). In the Smyd3 structure, however, the TPR domain encloses a large part of the substrate binding site, and together with the SET and post-SET domains forms a deep and narrow substrate binding pocket (Figure 2A and B). Detailed examination of the pocket shows that several acidic residues, including Glu192 and Asp241 of the SET domain and Asp332 of the TPR domain are located at the opening of the substrate binding pocket, and might be involved in histone binding (Figure 2B).Figure 2.


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)

Histone binding pocket. (A) Potential histone peptide binding site. Superposition of the Smyd3 and SET7/9 (PDB code 1O9S) structures was performed as in Figure 1B. Smyd3 is shown in a ribbon representation with the same color coding as in Figure 1A. For simplicity, for SET7/9 only the bound histone peptide is shown in a ribbon representation and colored in green and the side chain of the methyllysine of the histone peptide is shown with a ball-and-stick model and colored accordingly. The cofactor is also shown with a ball-and-stick model. (B) Electrostatic potential surface of the potential histone peptide binding pocket in Smyd3. The surface charge distribution is displayed as blue for positive, red for negative, and white for neutral. A close-up view of the pocket (middle panel) shows that several acidic patches are present at the opening of the binding pocket. Some of the acidic residues in these patches are labeled. The TPR domain forms part of the substrate binding pocket and removal of the TPR domain would leave an incomplete pocket (right panel). (C) HKMT activity assays of the wild-type Smyd3 and the mutants with truncation or mutations at the substrate binding pocket. The activities of the truncate with deletion of the C-terminal region (Δ277–428) and the mutants carrying one or two point mutations of the residues potentially involved in histone binding were determined. Activity is shown as the relative activity of the proteins normalized to that of the wild-type protein. The experiments were performed in triplicates and the error bars indicate the standard deviation.
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Related In: Results  -  Collection

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Figure 2: Histone binding pocket. (A) Potential histone peptide binding site. Superposition of the Smyd3 and SET7/9 (PDB code 1O9S) structures was performed as in Figure 1B. Smyd3 is shown in a ribbon representation with the same color coding as in Figure 1A. For simplicity, for SET7/9 only the bound histone peptide is shown in a ribbon representation and colored in green and the side chain of the methyllysine of the histone peptide is shown with a ball-and-stick model and colored accordingly. The cofactor is also shown with a ball-and-stick model. (B) Electrostatic potential surface of the potential histone peptide binding pocket in Smyd3. The surface charge distribution is displayed as blue for positive, red for negative, and white for neutral. A close-up view of the pocket (middle panel) shows that several acidic patches are present at the opening of the binding pocket. Some of the acidic residues in these patches are labeled. The TPR domain forms part of the substrate binding pocket and removal of the TPR domain would leave an incomplete pocket (right panel). (C) HKMT activity assays of the wild-type Smyd3 and the mutants with truncation or mutations at the substrate binding pocket. The activities of the truncate with deletion of the C-terminal region (Δ277–428) and the mutants carrying one or two point mutations of the residues potentially involved in histone binding were determined. Activity is shown as the relative activity of the proteins normalized to that of the wild-type protein. The experiments were performed in triplicates and the error bars indicate the standard deviation.
Mentions: Our attempts to obtain a structure of Smyd3 in complex with the histone peptide were unsuccessful. The Smyd3–AdoHcy structure was superposed to the structure of SET7/9 in complex with a methylated histone peptide (PDB code 1O9S) (25) (Figures 1B and 2A). In SET7/9, a narrow lysine channel is detected connecting the cofactor binding site and the histone peptide binding site (25). In the Smyd3–AdoHcy complex, a similar channel is present (see details later) and at one end of the channel AdoHcy binds at an almost identical position as in the SET7/9 complex (Figure 1B). Thus, we reason that the histone substrate of Smyd3 should bind at the other end of the channel as observed in SET7/9. In the SET7/9 structures, the histone peptide binding site is quite open as the N-terminal pre-SET domain is distant from the active site (21,25). In the Smyd3 structure, however, the TPR domain encloses a large part of the substrate binding site, and together with the SET and post-SET domains forms a deep and narrow substrate binding pocket (Figure 2A and B). Detailed examination of the pocket shows that several acidic residues, including Glu192 and Asp241 of the SET domain and Asp332 of the TPR domain are located at the opening of the substrate binding pocket, and might be involved in histone binding (Figure 2B).Figure 2.

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