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Functional redundancy between the transcriptional activation domains of E2A is mediated by binding to the KIX domain of CBP/p300.

Denis CM, Langelaan DN, Kirlin AC, Chitayat S, Munro K, Spencer HL, LeBrun DP, Smith SP - Nucleic Acids Res. (2014)

Bottom Line: The E2A gene is also involved in a chromosomal translocation that results in the leukemogenic oncoprotein E2A-PBX1.Mutagenesis uncovered a correspondence between the KIX-binding affinity of AD2 and transcriptional activation.Our findings suggest that redundancy between the two E2A activation domains with respect to transcriptional activation and oncogenic function is mediated by binding to the same surface of the KIX domain of CBP/p300.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, K7L 3N6, Canada.

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Related in: MedlinePlus

E2A AD2–1 residues critical to KIX domain recognition. (a) Backbone ribbon representation of a Modeller-generated structural model of the E2A AD2–1 peptide (orange) bound to the KIX domain (teal). The three α-helices of KIX are indicated, as are the N and C termini of both proteins. (b) The E2A AD2–1:KIX interaction surface from the model of the complex with stick representations of the side chains of residues that are potentially forming contacts. (c) Fluorescence anisotropy titrations of FITC-labelled E2A AD2–1 mutant synthetic peptides (•, AD2–1 wild-type; ○, AD2–1 Ala400Leu; ▾, AD2–1 His402Ala; ▵, AD2–1 Leu404Ala; ▪, AD2–1 Leu397Ala; □, Ile401Ala) informed by the model in (b) with recombinant KIX domain, where the curves are fitted to the average values of each titration point from three replicate experiments and error bars represent the standard error of each titration point among the replicate experiments. (d) The impact of E2A AD2–1 residue-specific substitutions listed in (c) on transcriptional activation of a Gal4-responsive luciferase reporter gene in HEK293T cells was tested using wild-type or the abovementioned mutant Gal4-E2A 1–483 constructs.
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Figure 6: E2A AD2–1 residues critical to KIX domain recognition. (a) Backbone ribbon representation of a Modeller-generated structural model of the E2A AD2–1 peptide (orange) bound to the KIX domain (teal). The three α-helices of KIX are indicated, as are the N and C termini of both proteins. (b) The E2A AD2–1:KIX interaction surface from the model of the complex with stick representations of the side chains of residues that are potentially forming contacts. (c) Fluorescence anisotropy titrations of FITC-labelled E2A AD2–1 mutant synthetic peptides (•, AD2–1 wild-type; ○, AD2–1 Ala400Leu; ▾, AD2–1 His402Ala; ▵, AD2–1 Leu404Ala; ▪, AD2–1 Leu397Ala; □, Ile401Ala) informed by the model in (b) with recombinant KIX domain, where the curves are fitted to the average values of each titration point from three replicate experiments and error bars represent the standard error of each titration point among the replicate experiments. (d) The impact of E2A AD2–1 residue-specific substitutions listed in (c) on transcriptional activation of a Gal4-responsive luciferase reporter gene in HEK293T cells was tested using wild-type or the abovementioned mutant Gal4-E2A 1–483 constructs.

Mentions: A structural model of the E2A AD2–1:KIX complex was generated using the E2A AD1-PCET:KIX complex structure (PDB: 2KWF; (32)) as a template in Modeller (47) to aid in deciphering the molecular basis of this interaction (Figure 6a and b). The model suggested that the E2A AD2–1:KIX interaction involved numerous hydrophobic interactions, particularly along one face of the helical E2A AD2–1 peptide that is buried in the hydrophobic cleft on the KIX domain (Figure 6b). The hydrophobic residues Leu397, Ala400 and Ile401 within the ϕ-x-x-ϕ-ϕ sequence of E2A AD2–1 were predicted to participate in extensive contacts with the KIX domain. These include interactions of Leu397 of E2A AD2–1 with non-polar side chains on the α2 and α3 helices of the KIX domain, Ala400 with non-polar side chains located on the α1 and α2 helices and Ile401 with non-polar side chains on the α3 helix of the KIX domain. Additionally, Leu404 was positioned to form hydrophobic interactions with non-polar residues within the L12 loop and the α3 helix. Overall, these results are consistent with the fluorescence anisotropy data, in which the KIXΔMybsite mutant bound E2A AD2–1 with wild-type affinity while the KIXΔPCETsite mutant had a significantly lower affinity for E2A AD2–1, and indicate that E2A AD2–1 binds to the surface on the KIX domain that is also bound by E2A AD1-PCET.


Functional redundancy between the transcriptional activation domains of E2A is mediated by binding to the KIX domain of CBP/p300.

Denis CM, Langelaan DN, Kirlin AC, Chitayat S, Munro K, Spencer HL, LeBrun DP, Smith SP - Nucleic Acids Res. (2014)

E2A AD2–1 residues critical to KIX domain recognition. (a) Backbone ribbon representation of a Modeller-generated structural model of the E2A AD2–1 peptide (orange) bound to the KIX domain (teal). The three α-helices of KIX are indicated, as are the N and C termini of both proteins. (b) The E2A AD2–1:KIX interaction surface from the model of the complex with stick representations of the side chains of residues that are potentially forming contacts. (c) Fluorescence anisotropy titrations of FITC-labelled E2A AD2–1 mutant synthetic peptides (•, AD2–1 wild-type; ○, AD2–1 Ala400Leu; ▾, AD2–1 His402Ala; ▵, AD2–1 Leu404Ala; ▪, AD2–1 Leu397Ala; □, Ile401Ala) informed by the model in (b) with recombinant KIX domain, where the curves are fitted to the average values of each titration point from three replicate experiments and error bars represent the standard error of each titration point among the replicate experiments. (d) The impact of E2A AD2–1 residue-specific substitutions listed in (c) on transcriptional activation of a Gal4-responsive luciferase reporter gene in HEK293T cells was tested using wild-type or the abovementioned mutant Gal4-E2A 1–483 constructs.
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Figure 6: E2A AD2–1 residues critical to KIX domain recognition. (a) Backbone ribbon representation of a Modeller-generated structural model of the E2A AD2–1 peptide (orange) bound to the KIX domain (teal). The three α-helices of KIX are indicated, as are the N and C termini of both proteins. (b) The E2A AD2–1:KIX interaction surface from the model of the complex with stick representations of the side chains of residues that are potentially forming contacts. (c) Fluorescence anisotropy titrations of FITC-labelled E2A AD2–1 mutant synthetic peptides (•, AD2–1 wild-type; ○, AD2–1 Ala400Leu; ▾, AD2–1 His402Ala; ▵, AD2–1 Leu404Ala; ▪, AD2–1 Leu397Ala; □, Ile401Ala) informed by the model in (b) with recombinant KIX domain, where the curves are fitted to the average values of each titration point from three replicate experiments and error bars represent the standard error of each titration point among the replicate experiments. (d) The impact of E2A AD2–1 residue-specific substitutions listed in (c) on transcriptional activation of a Gal4-responsive luciferase reporter gene in HEK293T cells was tested using wild-type or the abovementioned mutant Gal4-E2A 1–483 constructs.
Mentions: A structural model of the E2A AD2–1:KIX complex was generated using the E2A AD1-PCET:KIX complex structure (PDB: 2KWF; (32)) as a template in Modeller (47) to aid in deciphering the molecular basis of this interaction (Figure 6a and b). The model suggested that the E2A AD2–1:KIX interaction involved numerous hydrophobic interactions, particularly along one face of the helical E2A AD2–1 peptide that is buried in the hydrophobic cleft on the KIX domain (Figure 6b). The hydrophobic residues Leu397, Ala400 and Ile401 within the ϕ-x-x-ϕ-ϕ sequence of E2A AD2–1 were predicted to participate in extensive contacts with the KIX domain. These include interactions of Leu397 of E2A AD2–1 with non-polar side chains on the α2 and α3 helices of the KIX domain, Ala400 with non-polar side chains located on the α1 and α2 helices and Ile401 with non-polar side chains on the α3 helix of the KIX domain. Additionally, Leu404 was positioned to form hydrophobic interactions with non-polar residues within the L12 loop and the α3 helix. Overall, these results are consistent with the fluorescence anisotropy data, in which the KIXΔMybsite mutant bound E2A AD2–1 with wild-type affinity while the KIXΔPCETsite mutant had a significantly lower affinity for E2A AD2–1, and indicate that E2A AD2–1 binds to the surface on the KIX domain that is also bound by E2A AD1-PCET.

Bottom Line: The E2A gene is also involved in a chromosomal translocation that results in the leukemogenic oncoprotein E2A-PBX1.Mutagenesis uncovered a correspondence between the KIX-binding affinity of AD2 and transcriptional activation.Our findings suggest that redundancy between the two E2A activation domains with respect to transcriptional activation and oncogenic function is mediated by binding to the same surface of the KIX domain of CBP/p300.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, K7L 3N6, Canada.

Show MeSH
Related in: MedlinePlus