Elucidating the Signal Transduction Mechanism of the Blue-Light-Regulated Photoreceptor YtvA: From Photoactivation to Downstream Regulation
Elucidating the Signal Transduction Mechanism of the Blue-Light-Regulated Photoreceptor YtvA: From Photoactivation to Downstream Regulation
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DOI：
10.1021/acschembio.3c00722
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发表时间：
2024-02-22
影响因子：
4
通讯作者：
Tonge，Peter J.
中科院分区：
文献类型：
--
作者：
He，YongLe;Collado，Jinnette Tolentino;Tonge，Peter J.
来自枯草芽孢杆菌的蓝光光感受器 YtvA 具有 N 端黄素单核苷酸 (FMN) 结合光氧电压 (LOV) 结构域，该结构域与 C 端硫酸盐转运蛋白和抗 σ 因子拮抗剂 (STAS) 输出结构域融合。为了探究导致光激活的信号转导途径，将 STAS 结构域替换为组氨酸激酶，以便黄素的光激活可以与生物活性直接相关。 N94 是与 FMN C2=O 基团氢键结合的保守 Asn，被 Ala、Asp 和 Ser 残基取代，以探索该残基在触发激活输出域的结构动力学中的作用。飞秒至毫秒时间分辨多探针光谱与荧光偏振测定相结合表明，N94 和 C2=O 基团之间氢键的丧失使光激发引起的蛋白质结构变化解耦。此外，N94 的改变还使光激活状态下形成的 Cys-FMN 加合物的稳定性降低了约 25 倍。总的来说，这些研究揭示了 LOV β-支架中氢键网络在信号转导中的作用。
The blue-light photoreceptor YtvA fromBacillus subtilishas an N-terminal flavin mononucleotide (FMN)-binding light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate transporter and anti-σ factor antagonist (STAS) output domain. To interrogate the signal transduction pathway that leads to photoactivation, the STAS domain was replaced with a histidine kinase, so that photoexcitation of the flavin could be directly correlated with biological activity. N94, a conserved Asn that is hydrogen bonded to the FMN C2═O group, was replaced with Ala, Asp, and Ser residues to explore the role of this residue in triggering the structural dynamics that activate the output domain. Femtosecond to millisecond time-resolved multiple probe spectroscopy coupled with a fluorescence polarization assay revealed that the loss of the hydrogen bond between N94 and the C2═O group decoupled changes in the protein structure from photoexcitation. In addition, alterations in N94 also decreased the stability of the Cys-FMN adduct formed in the light-activated state by up to a factor of ∼25. Collectively, these studies shed light on the role of the hydrogen bonding network in the LOV β-scaffold in signal transduction.