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Structure of p300 bound to MEF2 on DNA reveals a mechanism of enhanceosome assembly.

He J, Ye J, Cai Y, Riquelme C, Liu JO, Liu X, Han A, Chen L - Nucleic Acids Res. (2011)

Bottom Line: Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously.These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein-protein interactions.Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.

View Article: PubMed Central - PubMed

Affiliation: MOE Key Laboratory for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

ABSTRACT
Transcription co-activators CBP and p300 are recruited by sequence-specific transcription factors to specific genomic loci to control gene expression. A highly conserved domain in CBP/p300, the TAZ2 domain, mediates direct interaction with a variety of transcription factors including the myocyte enhancer factor 2 (MEF2). Here we report the crystal structure of a ternary complex of the p300 TAZ2 domain bound to MEF2 on DNA at 2.2Å resolution. The structure reveals three MEF2:DNA complexes binding to different sites of the TAZ2 domain. Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously. These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein-protein interactions. Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.

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Structure of the p300 TAZ2 domain bound to MEF2 on DNA. (A) Overall structure in ribbon style. The MEF2 dimers are colored in yellow, magenta and blue, respectively; p300 is in green; the DNA backbone is shown in gold and its sequence is listed below. The secondary structural elements of one MEF2 dimer are labeled. (B) Protein binding interfaces: the proteins in the complex are shown in surface model and colored as in A. The three protein–protein interaction interfaces are labeled as IF-I, IF-II and IF-III. (C) Zoom-in view of the p300 TAZ2 domain. The four helices (α1–α4) and three zinc fingers (red spheres) are indicated. Part of the second zinc binding loop is disordered and labeled in a dashed line. (D) Superposition of the p300 TAZ2 domain in the MEF2-bound complex (green) with the unbound form (blue, 3IO2) (70).
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Figure 2: Structure of the p300 TAZ2 domain bound to MEF2 on DNA. (A) Overall structure in ribbon style. The MEF2 dimers are colored in yellow, magenta and blue, respectively; p300 is in green; the DNA backbone is shown in gold and its sequence is listed below. The secondary structural elements of one MEF2 dimer are labeled. (B) Protein binding interfaces: the proteins in the complex are shown in surface model and colored as in A. The three protein–protein interaction interfaces are labeled as IF-I, IF-II and IF-III. (C) Zoom-in view of the p300 TAZ2 domain. The four helices (α1–α4) and three zinc fingers (red spheres) are indicated. Part of the second zinc binding loop is disordered and labeled in a dashed line. (D) Superposition of the p300 TAZ2 domain in the MEF2-bound complex (green) with the unbound form (blue, 3IO2) (70).

Mentions: In order to analyze the details of p300:MEF2 interaction, we crystallized a complex between p300 (1726–1837) and MEF2A (1–95) on DNA. Our initial crystallization was hindered by the instability of p300 (1726–1837) due to the large number of cysteine residues in this region. Based on the NMR structure of the CBP TAZ2 domain (59), we mutated four cysteine residues (Cys1738, 1746, 1789 and 1790) that do not coordinate with zinc to serine. The cysteine-mutated p300 TAZ2 domain showed much improved stability and retained the same ability to bind MEF2 as the wild-type protein (data not shown). Using this engineered p300 (1726–1837) together with MEF2A (1–95) and a double stranded oligonucleotide containing the consensus MEF2 binding element (see Figure 2A below), we successfully crystallized the p300:MEF2:DNA complex. The crystals diffracted to 2.2Å. The statistics of data collection and structure refinement are summarized in Table 1.Figure 2.


Structure of p300 bound to MEF2 on DNA reveals a mechanism of enhanceosome assembly.

He J, Ye J, Cai Y, Riquelme C, Liu JO, Liu X, Han A, Chen L - Nucleic Acids Res. (2011)

Structure of the p300 TAZ2 domain bound to MEF2 on DNA. (A) Overall structure in ribbon style. The MEF2 dimers are colored in yellow, magenta and blue, respectively; p300 is in green; the DNA backbone is shown in gold and its sequence is listed below. The secondary structural elements of one MEF2 dimer are labeled. (B) Protein binding interfaces: the proteins in the complex are shown in surface model and colored as in A. The three protein–protein interaction interfaces are labeled as IF-I, IF-II and IF-III. (C) Zoom-in view of the p300 TAZ2 domain. The four helices (α1–α4) and three zinc fingers (red spheres) are indicated. Part of the second zinc binding loop is disordered and labeled in a dashed line. (D) Superposition of the p300 TAZ2 domain in the MEF2-bound complex (green) with the unbound form (blue, 3IO2) (70).
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Related In: Results  -  Collection

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Figure 2: Structure of the p300 TAZ2 domain bound to MEF2 on DNA. (A) Overall structure in ribbon style. The MEF2 dimers are colored in yellow, magenta and blue, respectively; p300 is in green; the DNA backbone is shown in gold and its sequence is listed below. The secondary structural elements of one MEF2 dimer are labeled. (B) Protein binding interfaces: the proteins in the complex are shown in surface model and colored as in A. The three protein–protein interaction interfaces are labeled as IF-I, IF-II and IF-III. (C) Zoom-in view of the p300 TAZ2 domain. The four helices (α1–α4) and three zinc fingers (red spheres) are indicated. Part of the second zinc binding loop is disordered and labeled in a dashed line. (D) Superposition of the p300 TAZ2 domain in the MEF2-bound complex (green) with the unbound form (blue, 3IO2) (70).
Mentions: In order to analyze the details of p300:MEF2 interaction, we crystallized a complex between p300 (1726–1837) and MEF2A (1–95) on DNA. Our initial crystallization was hindered by the instability of p300 (1726–1837) due to the large number of cysteine residues in this region. Based on the NMR structure of the CBP TAZ2 domain (59), we mutated four cysteine residues (Cys1738, 1746, 1789 and 1790) that do not coordinate with zinc to serine. The cysteine-mutated p300 TAZ2 domain showed much improved stability and retained the same ability to bind MEF2 as the wild-type protein (data not shown). Using this engineered p300 (1726–1837) together with MEF2A (1–95) and a double stranded oligonucleotide containing the consensus MEF2 binding element (see Figure 2A below), we successfully crystallized the p300:MEF2:DNA complex. The crystals diffracted to 2.2Å. The statistics of data collection and structure refinement are summarized in Table 1.Figure 2.

Bottom Line: Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously.These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein-protein interactions.Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.

View Article: PubMed Central - PubMed

Affiliation: MOE Key Laboratory for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

ABSTRACT
Transcription co-activators CBP and p300 are recruited by sequence-specific transcription factors to specific genomic loci to control gene expression. A highly conserved domain in CBP/p300, the TAZ2 domain, mediates direct interaction with a variety of transcription factors including the myocyte enhancer factor 2 (MEF2). Here we report the crystal structure of a ternary complex of the p300 TAZ2 domain bound to MEF2 on DNA at 2.2Å resolution. The structure reveals three MEF2:DNA complexes binding to different sites of the TAZ2 domain. Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously. These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein-protein interactions. Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.

Show MeSH
Related in: MedlinePlus