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Crystal structure of the human FOXO3a-DBD/DNA complex suggests the effects of post-translational modification.

Tsai KL, Sun YJ, Huang CY, Yang JY, Hung MC, Hsiao CD - Nucleic Acids Res. (2007)

Bottom Line: Because these post-translational modification sites are located within the C-terminal basic region of the FOXO DNA-binding domain (FOXO-DBD), it is possible that these post-translational modifications could alter the DNA-binding characteristics.Based on a unique structural feature in the C-terminal region and results from biochemical and mutational studies, our studies may explain how FOXO-DBD C-terminal phosphorylation by protein kinase B (PKB) or acetylation by cAMP-response element binding protein (CBP) can attenuate the DNA-binding activity and thereby reduce transcriptional activity of FOXO proteins.In addition, we demonstrate that the methyl groups of specific thymine bases within the consensus sequence are important for FOXO3a-DBD recognition of the consensus binding site.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.

ABSTRACT
FOXO3a is a transcription factor of the FOXO family. The FOXO proteins participate in multiple signaling pathways, and their transcriptional activity is regulated by several post-translational mechanisms, including phosphorylation, acetylation and ubiquitination. Because these post-translational modification sites are located within the C-terminal basic region of the FOXO DNA-binding domain (FOXO-DBD), it is possible that these post-translational modifications could alter the DNA-binding characteristics. To understand how FOXO mediate transcriptional activity, we report here the 2.7 A crystal structure of the DNA-binding domain of FOXO3a (FOXO3a-DBD) bound to a 13-bp DNA duplex containing a FOXO consensus binding sequence (GTAAACA). Based on a unique structural feature in the C-terminal region and results from biochemical and mutational studies, our studies may explain how FOXO-DBD C-terminal phosphorylation by protein kinase B (PKB) or acetylation by cAMP-response element binding protein (CBP) can attenuate the DNA-binding activity and thereby reduce transcriptional activity of FOXO proteins. In addition, we demonstrate that the methyl groups of specific thymine bases within the consensus sequence are important for FOXO3a-DBD recognition of the consensus binding site.

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DNA-binding properties of FOXO3a-DBD proteins examined by fluorescence anisotropy assay. Wild-type FOXO3a-DBD158–253, K242A, K245A, S253D, RRR/AAA and FOXO3a-DBD158–240 were diluted and incubated with a fluorescein-labeled DNA at 25°C in 20 mM Tris–HCl, pH 7.6, NaCl 50 mM, DTT 1 mM. The anisotropy values were measured after 30 min incubation.
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Figure 7: DNA-binding properties of FOXO3a-DBD proteins examined by fluorescence anisotropy assay. Wild-type FOXO3a-DBD158–253, K242A, K245A, S253D, RRR/AAA and FOXO3a-DBD158–240 were diluted and incubated with a fluorescein-labeled DNA at 25°C in 20 mM Tris–HCl, pH 7.6, NaCl 50 mM, DTT 1 mM. The anisotropy values were measured after 30 min incubation.

Mentions: To compare the DNA-binding affinity of the FOXO3a-DBD proteins, we performed fluorescence anisotropy assay to measure the binding ability of these mutant proteins using a fluorescently labeled DNA containing the FOXO consensus binding sequence. As shown in Figure 7, we observed that the K24A and K245A mutants are defective in binding DNA compared to wild-type FOXO3a-DBD. The wild-type FOXO3a-DBD bound to DNA with an apparent Kd of 295 ± 26 nM (Table 2). However, the apparent Kd value for K242A and K245A mutants is 399 ± 25 nM and 511 ± 63 nM, respectively. These data support the results of gel shift studies that substitutions of K242 and K245 for alanine reduced the DNA-binding affinity of FOXO3a-DBD. In the structure, residue Ser253 interacted with phosphate group of DNA. To further study the role of PKB-induced phosphorylation at Ser253, we created a S253D mutant to mimic the phosphorylated state. The result showed that S253D mutant caused a decrease on its DNA-binding ability (Kd = 511 ± 63 nM), indicating that phosphorylation at Ser253 influences the stability of protein–DNA interaction. This effect is also consistent with the result that S256D of FOXO1 (Ser253 in FOXO3a) led a decrease in DNA-binding affinity (52). In addition, substitutions of the three basic residues (Arg248 ∼ 250) with alanine also significantly impaired DNA-binding affinity with an apparent Kd of 1178 ± 168 nM. Furthermore, the truncation of C-terminus at the FOXO3a-DBD also caused a 5-fold decrease for DNA-binding (Kd = 1492 ± 197 nM), indicating that the C-terminus of FOXO3a-DBD is important for DNA binding.Table 2.


Crystal structure of the human FOXO3a-DBD/DNA complex suggests the effects of post-translational modification.

Tsai KL, Sun YJ, Huang CY, Yang JY, Hung MC, Hsiao CD - Nucleic Acids Res. (2007)

DNA-binding properties of FOXO3a-DBD proteins examined by fluorescence anisotropy assay. Wild-type FOXO3a-DBD158–253, K242A, K245A, S253D, RRR/AAA and FOXO3a-DBD158–240 were diluted and incubated with a fluorescein-labeled DNA at 25°C in 20 mM Tris–HCl, pH 7.6, NaCl 50 mM, DTT 1 mM. The anisotropy values were measured after 30 min incubation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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Figure 7: DNA-binding properties of FOXO3a-DBD proteins examined by fluorescence anisotropy assay. Wild-type FOXO3a-DBD158–253, K242A, K245A, S253D, RRR/AAA and FOXO3a-DBD158–240 were diluted and incubated with a fluorescein-labeled DNA at 25°C in 20 mM Tris–HCl, pH 7.6, NaCl 50 mM, DTT 1 mM. The anisotropy values were measured after 30 min incubation.
Mentions: To compare the DNA-binding affinity of the FOXO3a-DBD proteins, we performed fluorescence anisotropy assay to measure the binding ability of these mutant proteins using a fluorescently labeled DNA containing the FOXO consensus binding sequence. As shown in Figure 7, we observed that the K24A and K245A mutants are defective in binding DNA compared to wild-type FOXO3a-DBD. The wild-type FOXO3a-DBD bound to DNA with an apparent Kd of 295 ± 26 nM (Table 2). However, the apparent Kd value for K242A and K245A mutants is 399 ± 25 nM and 511 ± 63 nM, respectively. These data support the results of gel shift studies that substitutions of K242 and K245 for alanine reduced the DNA-binding affinity of FOXO3a-DBD. In the structure, residue Ser253 interacted with phosphate group of DNA. To further study the role of PKB-induced phosphorylation at Ser253, we created a S253D mutant to mimic the phosphorylated state. The result showed that S253D mutant caused a decrease on its DNA-binding ability (Kd = 511 ± 63 nM), indicating that phosphorylation at Ser253 influences the stability of protein–DNA interaction. This effect is also consistent with the result that S256D of FOXO1 (Ser253 in FOXO3a) led a decrease in DNA-binding affinity (52). In addition, substitutions of the three basic residues (Arg248 ∼ 250) with alanine also significantly impaired DNA-binding affinity with an apparent Kd of 1178 ± 168 nM. Furthermore, the truncation of C-terminus at the FOXO3a-DBD also caused a 5-fold decrease for DNA-binding (Kd = 1492 ± 197 nM), indicating that the C-terminus of FOXO3a-DBD is important for DNA binding.Table 2.

Bottom Line: Because these post-translational modification sites are located within the C-terminal basic region of the FOXO DNA-binding domain (FOXO-DBD), it is possible that these post-translational modifications could alter the DNA-binding characteristics.Based on a unique structural feature in the C-terminal region and results from biochemical and mutational studies, our studies may explain how FOXO-DBD C-terminal phosphorylation by protein kinase B (PKB) or acetylation by cAMP-response element binding protein (CBP) can attenuate the DNA-binding activity and thereby reduce transcriptional activity of FOXO proteins.In addition, we demonstrate that the methyl groups of specific thymine bases within the consensus sequence are important for FOXO3a-DBD recognition of the consensus binding site.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.

ABSTRACT
FOXO3a is a transcription factor of the FOXO family. The FOXO proteins participate in multiple signaling pathways, and their transcriptional activity is regulated by several post-translational mechanisms, including phosphorylation, acetylation and ubiquitination. Because these post-translational modification sites are located within the C-terminal basic region of the FOXO DNA-binding domain (FOXO-DBD), it is possible that these post-translational modifications could alter the DNA-binding characteristics. To understand how FOXO mediate transcriptional activity, we report here the 2.7 A crystal structure of the DNA-binding domain of FOXO3a (FOXO3a-DBD) bound to a 13-bp DNA duplex containing a FOXO consensus binding sequence (GTAAACA). Based on a unique structural feature in the C-terminal region and results from biochemical and mutational studies, our studies may explain how FOXO-DBD C-terminal phosphorylation by protein kinase B (PKB) or acetylation by cAMP-response element binding protein (CBP) can attenuate the DNA-binding activity and thereby reduce transcriptional activity of FOXO proteins. In addition, we demonstrate that the methyl groups of specific thymine bases within the consensus sequence are important for FOXO3a-DBD recognition of the consensus binding site.

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