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<p>The transcription machinery is assembled via interactions of DNA-bound transcriptional activators and coactivators. When the eukaryotic RNA polymerase II complex is formed, cAMP-regulated transcription factor (CREB) binding protein (CBP) acts as a general coactivator bridging the transcriptional apparatus. Forkhead box protein O4 (FOXO4), a transcription factor, has been reported to bind to the KIX domain of CBP (CBP-KIX). Although the CR3 of FOXO4 (FOXO4-CR3) binds as expected to the MLL and c-Myb sites of CBP-KIX, its substantially higher affinity for CBP, compared to its homolog FOXO3a, cannot be explained by a single conserved ΦXXΦΦ binding motif. Here, we found that a second ΦXXΦΦ motif in FOXO4-CR3 provides an additional point of contact for CBP-KIX. Isothermal titration calorimetry and chemical shift perturbation analyses revealed a difference in binding affinity and confirmed that different binding patterns occur at the two hydrophobic pockets of CBP-KIX. Increased helicity of FOXO4-CR3 upon KIX MLL site binding was demonstrated by circular dichroism and Cα chemical shifts. Paramagnetic relaxation enhancement and docking simulations suggested FOXO4-CR3 orientation is not restrained in the KIX-CR3 complex. Our study provides information about the unique binding properties of FOXO4-CR3 and CBP-KIX, expanding our understanding of CBP recruitment via KIX-transactivation domain binding.</p><p></p>

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Recognition of two hydrophobic pockets in the KIX domain of CBP by FOXO4 transactivation domain

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月11日

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  • Jeongbeen Heo,
  • Kyoung-Seok Ryu,
  • Chin-Ju Park

摘要

The transcription machinery is assembled via interactions of DNA-bound transcriptional activators and coactivators. When the eukaryotic RNA polymerase II complex is formed, cAMP-regulated transcription factor (CREB) binding protein (CBP) acts as a general coactivator bridging the transcriptional apparatus. Forkhead box protein O4 (FOXO4), a transcription factor, has been reported to bind to the KIX domain of CBP (CBP-KIX). Although the CR3 of FOXO4 (FOXO4-CR3) binds as expected to the MLL and c-Myb sites of CBP-KIX, its substantially higher affinity for CBP, compared to its homolog FOXO3a, cannot be explained by a single conserved ΦXXΦΦ binding motif. Here, we found that a second ΦXXΦΦ motif in FOXO4-CR3 provides an additional point of contact for CBP-KIX. Isothermal titration calorimetry and chemical shift perturbation analyses revealed a difference in binding affinity and confirmed that different binding patterns occur at the two hydrophobic pockets of CBP-KIX. Increased helicity of FOXO4-CR3 upon KIX MLL site binding was demonstrated by circular dichroism and Cα chemical shifts. Paramagnetic relaxation enhancement and docking simulations suggested FOXO4-CR3 orientation is not restrained in the KIX-CR3 complex. Our study provides information about the unique binding properties of FOXO4-CR3 and CBP-KIX, expanding our understanding of CBP recruitment via KIX-transactivation domain binding.