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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

Modulation of the HKMT activity of Smyd3 via DNA binding of the MYND Domain. (A) Electrostatic potential surface of the MYND domain with the negative charge in red and the positive charge in blue. All the positively charged residues are labeled. (B) Analysis of the interaction between the wild-type and mutant Smyd3 proteins and the potential target DNA with gel shift assays. The wild-type (WT) Smyd3 and the R66E and E192A mutants were loaded to 1 µg DNA with different molar ratios as indicated. DNA binding of the wild-type and mutant Smyd3 were analyzed with agarose gel electrophoresis. (C) HKMT assays of the wild-type and R66E mutant Smyd3 in the presence and absence of the potential target DNA. The HKMT activity of the wild-type but not the mutant Smyd3 is stimulated when the potential target DNA is supplemented in the HKMT reaction system. (D) Sequence alignment of the MYND domain among all human Smyd proteins. The sequence number and secondary structure elements of Smyd3 are marked. The invariant residues across these proteins are denoted with filled red boxes, and the highly conserved ones in open boxes. The residues conserved in Smyd1-4 but not Smyd5 are denoted with asterisks.
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Figure 5: Modulation of the HKMT activity of Smyd3 via DNA binding of the MYND Domain. (A) Electrostatic potential surface of the MYND domain with the negative charge in red and the positive charge in blue. All the positively charged residues are labeled. (B) Analysis of the interaction between the wild-type and mutant Smyd3 proteins and the potential target DNA with gel shift assays. The wild-type (WT) Smyd3 and the R66E and E192A mutants were loaded to 1 µg DNA with different molar ratios as indicated. DNA binding of the wild-type and mutant Smyd3 were analyzed with agarose gel electrophoresis. (C) HKMT assays of the wild-type and R66E mutant Smyd3 in the presence and absence of the potential target DNA. The HKMT activity of the wild-type but not the mutant Smyd3 is stimulated when the potential target DNA is supplemented in the HKMT reaction system. (D) Sequence alignment of the MYND domain among all human Smyd proteins. The sequence number and secondary structure elements of Smyd3 are marked. The invariant residues across these proteins are denoted with filled red boxes, and the highly conserved ones in open boxes. The residues conserved in Smyd1-4 but not Smyd5 are denoted with asterisks.

Mentions: It has been indicated that the MYND domain of AML1/ETO binds the PPPL1 motif of retinoid and thyroid hormone receptor SMRT corepressor (50). In the structure of the MYND domain of AML1/ETO which is fused to the PPPL1 motif of SMRT, the conformation of the MYND domain is similar to that of the unbound form, and the three residues Ser675, Gln688 and Trp692 of the MYND domain located in a hydrophobic pocket interact with the PPPLI motif of the SMRT peptide (50). In the MYND domain of Smyd3, the equivalent residues (Ser63, Glu76 and Trp80, respectively) are conserved. However, the electrostatic properties of the residues surrounding the hydrophobic pocket exhibit substantial differences, with the replacement of acidic residues (Glu672 and Glu692) of AML1/ETO with hydrophobic residues (Met60 and Pro81) and substitution of residues Thr673 and His689 with highly basic residues (Arg61 and Lys77) (Figure 5A). With the presence of additional Lys and Arg residues, the surface of the MYND domain in Smyd3 is largely positively charged (Figure 5A), which is in agreement with its potential role in the binding of specific DNA sequences such as 5′-CCCTCC-3′ and further in transcriptional regulation of the targets including Nkx2.8 (6).Figure 5.


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)

Modulation of the HKMT activity of Smyd3 via DNA binding of the MYND Domain. (A) Electrostatic potential surface of the MYND domain with the negative charge in red and the positive charge in blue. All the positively charged residues are labeled. (B) Analysis of the interaction between the wild-type and mutant Smyd3 proteins and the potential target DNA with gel shift assays. The wild-type (WT) Smyd3 and the R66E and E192A mutants were loaded to 1 µg DNA with different molar ratios as indicated. DNA binding of the wild-type and mutant Smyd3 were analyzed with agarose gel electrophoresis. (C) HKMT assays of the wild-type and R66E mutant Smyd3 in the presence and absence of the potential target DNA. The HKMT activity of the wild-type but not the mutant Smyd3 is stimulated when the potential target DNA is supplemented in the HKMT reaction system. (D) Sequence alignment of the MYND domain among all human Smyd proteins. The sequence number and secondary structure elements of Smyd3 are marked. The invariant residues across these proteins are denoted with filled red boxes, and the highly conserved ones in open boxes. The residues conserved in Smyd1-4 but not Smyd5 are denoted with asterisks.
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Related In: Results  -  Collection

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Figure 5: Modulation of the HKMT activity of Smyd3 via DNA binding of the MYND Domain. (A) Electrostatic potential surface of the MYND domain with the negative charge in red and the positive charge in blue. All the positively charged residues are labeled. (B) Analysis of the interaction between the wild-type and mutant Smyd3 proteins and the potential target DNA with gel shift assays. The wild-type (WT) Smyd3 and the R66E and E192A mutants were loaded to 1 µg DNA with different molar ratios as indicated. DNA binding of the wild-type and mutant Smyd3 were analyzed with agarose gel electrophoresis. (C) HKMT assays of the wild-type and R66E mutant Smyd3 in the presence and absence of the potential target DNA. The HKMT activity of the wild-type but not the mutant Smyd3 is stimulated when the potential target DNA is supplemented in the HKMT reaction system. (D) Sequence alignment of the MYND domain among all human Smyd proteins. The sequence number and secondary structure elements of Smyd3 are marked. The invariant residues across these proteins are denoted with filled red boxes, and the highly conserved ones in open boxes. The residues conserved in Smyd1-4 but not Smyd5 are denoted with asterisks.
Mentions: It has been indicated that the MYND domain of AML1/ETO binds the PPPL1 motif of retinoid and thyroid hormone receptor SMRT corepressor (50). In the structure of the MYND domain of AML1/ETO which is fused to the PPPL1 motif of SMRT, the conformation of the MYND domain is similar to that of the unbound form, and the three residues Ser675, Gln688 and Trp692 of the MYND domain located in a hydrophobic pocket interact with the PPPLI motif of the SMRT peptide (50). In the MYND domain of Smyd3, the equivalent residues (Ser63, Glu76 and Trp80, respectively) are conserved. However, the electrostatic properties of the residues surrounding the hydrophobic pocket exhibit substantial differences, with the replacement of acidic residues (Glu672 and Glu692) of AML1/ETO with hydrophobic residues (Met60 and Pro81) and substitution of residues Thr673 and His689 with highly basic residues (Arg61 and Lys77) (Figure 5A). With the presence of additional Lys and Arg residues, the surface of the MYND domain in Smyd3 is largely positively charged (Figure 5A), which is in agreement with its potential role in the binding of specific DNA sequences such as 5′-CCCTCC-3′ and further in transcriptional regulation of the targets including Nkx2.8 (6).Figure 5.

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