Regulatory Implications of Structural Changes in Tyr201 of the Oxygen Sensor Protein FixL
Regulatory Implications of Structural Changes in Tyr201 of the Oxygen Sensor Protein FixL
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氧传感器蛋白 FixL 的 Tyr201 结构变化的监管意义
DOI：
10.1021/acs.biochem.6b00405
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发表时间：
2016
期刊：
影响因子：
2.9
通讯作者：
and Yasuhisa Mizutani
中科院分区：
文献类型：
--
作者：
Takeo Yamawaki;Haruto Ishikawa;Misao Mizuno;Hiro Nakamura;Yoshitsugu Shiro;and Yasuhisa Mizutani
FixL 是一种基于血红素的氧敏感组氨酸激酶，可在缺氧条件下诱导固氮基因的表达。 FixL 传感器结构域中血红素铁的氧解离引发蛋白质构象变化，这些变化传递到组氨酸激酶结构域，激活自磷酸化活性。相反，氧结合会抑制 FixL 激酶活性。为了理解变构转导机制，阐明氧解离时蛋白质结构发生的变化至关重要。我们测量了 FixL 及其突变体的脱氧、氧和一碳氧形式的紫外共振拉曼光谱，以检查氧解离时蛋白质结构的变化。观察到的光谱变化表明 Tyr201 及其邻近残基在氧解离时发生结构变化。激酶测定表明，Tyr201 的取代显着降低了氧结合时激酶活性的抑制。这些数据意味着由氧解离引起的 Tyr201 氢键的减弱对于抑制激酶活性至关重要。我们还观察到 FixL 氧解离后激酶结构域中 Tyr 残基的光谱变化，这是首次观察到 FixL 激酶结构域中氧依赖性结构变化。观察到的结构变化支持我们之前提出的 FixL 变构转导途径 [Yano, S., Ishikawa, H., Mizuno, M., Nakamura, H., Shiro, Y., and Mizutani, Y. (2013) J. Phys.化学。 B 117, 15786−15791]。
FixL is a heme-based oxygen-sensing histidine kinase that induces the expression of nitrogen fixation genes under hypoxic conditions. Oxygen dissociation from heme iron in the sensor domain of FixL initiates protein conformational changes that are transmitted to the histidine kinase domain, activating autophosphorylation activity. Conversely, oxygen binding inhibits FixL kinase activity. It is essential to elucidate the changes that occur in the protein structure upon this oxygen dissociation for understanding of the allosteric transduction mechanism. We measured ultraviolet resonance Raman spectra of FixL and its mutants for deoxy, oxy, and carbonmonoxy forms to examine the changes in protein structure upon oxygen dissociation. The observed spectral changes indicated that Tyr201 and its neighboring residues undergo structural changes upon oxygen dissociation. Kinase assays showed that substitution of Tyr201 significantly decreased the inhibition of kinase activity upon oxygen binding. These data mean that weakening of the hydrogen bond of Tyr201 that is induced by oxygen dissociation is essential for inhibition of kinase activity. We also observed spectral changes in Tyr residues in the kinase domain upon oxygen dissociation from FixL, which is the first observation of oxygen-dependent structural changes in the kinase domain of FixL. The observed structural changes support the allosteric transduction pathway of FixL which we proposed previously [Yano, S., Ishikawa, H., Mizuno, M., Nakamura, H., Shiro, Y., and Mizutani, Y. (2013) J. Phys. Chem. B 117, 15786−15791].