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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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Electrophoretic mobility shift assay (EMSA). (A) EMSA was performed with wild-type, mutated and FOXO3a-DBD158–240 and a 32P-labeled oligonucleotide probe containing the FOXO consensus DNA-binding sequence, GTAAACA. In the control lane, only 32P-labeled DNA was used. Free probe is indicated at the bottom of the gel. (B) DNA-binding affinity of wild-type, mutant and FOXO3a-DBD158–240 with the insulin response sequence (IRS), CAAAACA. (C) EMSA of wild-type FOXO3a-DBD binding to oligonucleotides containing substitutions within the FOXO consensus sequence. Lane 1, 32P-labeled DNA only; lanes 2–7, wild-type sequence or sequences substituted with U at positions 2, 3, 4, 5 or 7, respectively. The nucleotide sequence of each substitution site is shown on the right-hand side of Figure 6C. The final FOXO3a-DBD concentration in lanes 2–7 was 800 nM. The extent of FOXO3a-DBD relative binding to DNA (indicated below each lane) was quantified using a PhosphorImager (Molecular Dynamics). Lane 1 is a control, with no added protein. Positions of free DNA and the protein–DNA complex are indicated on the left.
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Figure 6: Electrophoretic mobility shift assay (EMSA). (A) EMSA was performed with wild-type, mutated and FOXO3a-DBD158–240 and a 32P-labeled oligonucleotide probe containing the FOXO consensus DNA-binding sequence, GTAAACA. In the control lane, only 32P-labeled DNA was used. Free probe is indicated at the bottom of the gel. (B) DNA-binding affinity of wild-type, mutant and FOXO3a-DBD158–240 with the insulin response sequence (IRS), CAAAACA. (C) EMSA of wild-type FOXO3a-DBD binding to oligonucleotides containing substitutions within the FOXO consensus sequence. Lane 1, 32P-labeled DNA only; lanes 2–7, wild-type sequence or sequences substituted with U at positions 2, 3, 4, 5 or 7, respectively. The nucleotide sequence of each substitution site is shown on the right-hand side of Figure 6C. The final FOXO3a-DBD concentration in lanes 2–7 was 800 nM. The extent of FOXO3a-DBD relative binding to DNA (indicated below each lane) was quantified using a PhosphorImager (Molecular Dynamics). Lane 1 is a control, with no added protein. Positions of free DNA and the protein–DNA complex are indicated on the left.

Mentions: To further characterize the roles of the residues involved in protein–DNA interactions in the FOXO3a-DBD/DNA structure, we studied the effect of various mutations on the ability of FOXO3a-DBD to bind to a DNA duplex containing either the FOXO consensus binding sequence 5′-GTAAACA-3′ or insulin response sequence (IRS) 5′-CAAAATA-3′ (50,51). Gel shift studies (Figure 6A and B) revealed that the FOXO3a-DBD158–240 lacking the C-terminal region almost lost the ability to bind DNA, indicating that the C-terminal region of FOXO3a-DBD contributes significantly to the formation of a stable protein–DNA complex. Substitutions of the three basic residues (Arg248 ∼ 250) with alanine also substantially diminished DNA binding. Because Lys242 and Lys245 are located in the C-terminus of FOXO3a-DBD, and Lys245 interacted with the phosphate group, we mutated these Lys residues to alanine and examined their effect on DNA binding. The K245A mutation reduced the DNA-binding ability more than the K242A mutation, consistent with the observed direct interaction between Lys245 and the DNA in the crystal structure. Although Lys242 did not contact the DNA, the K242A mutant showed a slight decrease in DNA affinity. We hypothesize that the K242A mutant impaired DNA binding by destabilizing the C-terminal region. We also examined the effect on DNA binding with the double mutant, K242A/K245A. Interestingly, DNA binding in this double mutant was dramatically reduced (Figure 6A and B), suggesting that K242 and K245 might have a synergistic effect on DNA binding.Figure 6.


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)

Electrophoretic mobility shift assay (EMSA). (A) EMSA was performed with wild-type, mutated and FOXO3a-DBD158–240 and a 32P-labeled oligonucleotide probe containing the FOXO consensus DNA-binding sequence, GTAAACA. In the control lane, only 32P-labeled DNA was used. Free probe is indicated at the bottom of the gel. (B) DNA-binding affinity of wild-type, mutant and FOXO3a-DBD158–240 with the insulin response sequence (IRS), CAAAACA. (C) EMSA of wild-type FOXO3a-DBD binding to oligonucleotides containing substitutions within the FOXO consensus sequence. Lane 1, 32P-labeled DNA only; lanes 2–7, wild-type sequence or sequences substituted with U at positions 2, 3, 4, 5 or 7, respectively. The nucleotide sequence of each substitution site is shown on the right-hand side of Figure 6C. The final FOXO3a-DBD concentration in lanes 2–7 was 800 nM. The extent of FOXO3a-DBD relative binding to DNA (indicated below each lane) was quantified using a PhosphorImager (Molecular Dynamics). Lane 1 is a control, with no added protein. Positions of free DNA and the protein–DNA complex are indicated on the left.
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Figure 6: Electrophoretic mobility shift assay (EMSA). (A) EMSA was performed with wild-type, mutated and FOXO3a-DBD158–240 and a 32P-labeled oligonucleotide probe containing the FOXO consensus DNA-binding sequence, GTAAACA. In the control lane, only 32P-labeled DNA was used. Free probe is indicated at the bottom of the gel. (B) DNA-binding affinity of wild-type, mutant and FOXO3a-DBD158–240 with the insulin response sequence (IRS), CAAAACA. (C) EMSA of wild-type FOXO3a-DBD binding to oligonucleotides containing substitutions within the FOXO consensus sequence. Lane 1, 32P-labeled DNA only; lanes 2–7, wild-type sequence or sequences substituted with U at positions 2, 3, 4, 5 or 7, respectively. The nucleotide sequence of each substitution site is shown on the right-hand side of Figure 6C. The final FOXO3a-DBD concentration in lanes 2–7 was 800 nM. The extent of FOXO3a-DBD relative binding to DNA (indicated below each lane) was quantified using a PhosphorImager (Molecular Dynamics). Lane 1 is a control, with no added protein. Positions of free DNA and the protein–DNA complex are indicated on the left.
Mentions: To further characterize the roles of the residues involved in protein–DNA interactions in the FOXO3a-DBD/DNA structure, we studied the effect of various mutations on the ability of FOXO3a-DBD to bind to a DNA duplex containing either the FOXO consensus binding sequence 5′-GTAAACA-3′ or insulin response sequence (IRS) 5′-CAAAATA-3′ (50,51). Gel shift studies (Figure 6A and B) revealed that the FOXO3a-DBD158–240 lacking the C-terminal region almost lost the ability to bind DNA, indicating that the C-terminal region of FOXO3a-DBD contributes significantly to the formation of a stable protein–DNA complex. Substitutions of the three basic residues (Arg248 ∼ 250) with alanine also substantially diminished DNA binding. Because Lys242 and Lys245 are located in the C-terminus of FOXO3a-DBD, and Lys245 interacted with the phosphate group, we mutated these Lys residues to alanine and examined their effect on DNA binding. The K245A mutation reduced the DNA-binding ability more than the K242A mutation, consistent with the observed direct interaction between Lys245 and the DNA in the crystal structure. Although Lys242 did not contact the DNA, the K242A mutant showed a slight decrease in DNA affinity. We hypothesize that the K242A mutant impaired DNA binding by destabilizing the C-terminal region. We also examined the effect on DNA binding with the double mutant, K242A/K245A. Interestingly, DNA binding in this double mutant was dramatically reduced (Figure 6A and B), suggesting that K242 and K245 might have a synergistic effect on DNA binding.Figure 6.

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.

Show MeSH