Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
Graduate School of Integrated Sciences for Life, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan.
Biosci Rep. 2023 Jul 26;43(7). doi: 10.1042/BSR20230846.
The general transcription factor TFIIH is a multi-subunit complex involved in transcription, DNA repair, and cell cycle in eukaryotes. In the human p62 subunit and the budding yeast Saccharomyces cerevisiae Tfb1 subunit of TFIIH, the pleckstrin homology (PH) domain (hPH/scPH) recruits TFIIH to transcription-start and DNA-damage sites by interacting with an acidic intrinsically disordered region in transcription and repair factors. Whereas metazoan PH domains are highly conserved and adopt a similar structure, fungal PH domains are divergent and only the scPH structure is available. Here, we have determined the structure of the PH domain from Tfb1 of fission yeast Schizosaccharomyces pombe (spPH) by NMR. spPH holds an architecture, including the core and external backbone structures, that is closer to hPH than to scPH despite having higher amino acid sequence identity to scPH. In addition, the predicted target-binding site of spPH shares more amino acid similarity with scPH, but spPH contains several key residues identified in hPH as required for specific binding. Using chemical shift perturbation, we have identified binding modes of spPH to spTfa1, a homologue of hTFIIEα, and to spRhp41, a homologue of the repair factors hXPC and scRad4. Both spTfa1 and spRhp41 bind to a similar but distinct surface of spPH by modes that differ from those of target proteins binding to hPH and scPH, revealing that the PH domain of TFIIH interacts with its target proteins in a polymorphic manner in Metazoa, and budding and fission yeasts.
一般转录因子 TFIIH 是一种多亚基复合物，参与真核生物的转录、DNA 修复和细胞周期。在人类 p62 亚基和芽殖酵母酿酒酵母 Tfb1 亚基的 TFIIH 中，pleckstrin 同源（PH）结构域（hPH/scPH）通过与转录和修复因子中的酸性无序结构域相互作用，将 TFIIH 募集到转录起始和 DNA 损伤位点。虽然后生动物的 PH 结构域高度保守且采用相似的结构，但真菌的 PH 结构域是不同的，只有 scPH 结构可用。在这里，我们通过 NMR 确定了裂殖酵母 Schizosaccharomyces pombe（spPH）Tfb1 的 PH 结构域的结构。尽管 spPH 与 scPH 的氨基酸序列同一性更高，但它具有更接近 hPH 的结构，包括核心和外部骨架结构。此外，spPH 的预测靶标结合位点与 scPH 共享更多的氨基酸相似性，但 spPH 包含在 hPH 中确定的几个关键残基，这些残基是特异性结合所必需的。通过化学位移扰动，我们确定了 spPH 与 spTfa1（hTFIIEα 的同源物）和 spRhp41（修复因子 hXPC 和 scRad4 的同源物）的结合模式。spTfa1 和 spRhp41 通过与 hPH 和 scPH 结合的靶蛋白不同的模式结合到 spPH 的相似但不同的表面，这表明 TFIIH 的 PH 结构域以多态方式与后生动物、芽殖和裂殖酵母的靶蛋白相互作用。
