Structural Characteristics of the Redox-sensing Coiled Coil in the Voltage-gated H+ Channel
Structural Characteristics of the Redox-sensing Coiled Coil in the Voltage-gated H+ Channel
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DOI：
10.1074/jbc.m113.459024
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发表时间：
2013-06-21
影响因子：
4.8
通讯作者：
Okamura, Yasushi
中科院分区：
文献类型：
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作者：
Fujiwara, Yuichiro;Takeshita, Kohei;Okamura, Yasushi
氧化是一种重要的生化防御机制，但它也会引起毒性，因此必须严格控制氧化。在中性粒细胞的吞噬活动中，电压门控H+(Hv)通道是对抗入侵细菌产生活性氧的关键调节因子。Hv通道的细胞质结构域形成支撑二聚化功能单元的二聚体卷曲线圈。重要的是，在螺旋线圈核心的排列中，保守的半胱氨酸残基形成了潜在的亚基间二硫键。在这项研究中，我们解决了螺旋结构域在还原、氧化和突变(Cys-&Gt；Ser)状态下的晶体结构。晶体结构表明，根据氧化还原条件，一对半胱氨酸残基形成亚基间二硫键。圆二色谱分析表明，二硫键提高了卷曲蛋白的热稳定性。我们还发现，两个硫醇修饰剂分子能够以氧化还原依赖的方式与半胱氨酸结合，而不会破坏二聚体螺旋螺旋组装。因此，Hv通道中细胞质螺旋线圈结构域的生化特性依赖于氧化还原条件，这可能在吞噬小体的氧化还原感觉中发挥作用。
Oxidation is an important biochemical defense mechanism, but it also elicits toxicity; therefore, oxidation must be under strict control. In phagocytotic events in neutrophils, the voltage-gated H+ (Hv) channel is a key regulator of the production of reactive oxygen species against invading bacteria. The cytoplasmic domain of the Hv channel forms a dimeric coiled coil underpinning a dimerized functional unit. Importantly, in the alignment of the coiled-coil core, a conserved cysteine residue forms a potential intersubunit disulfide bond. In this study, we solved the crystal structures of the coiled-coil domain in reduced, oxidized, and mutated (Cys -> Ser) states. The crystal structures indicate that a pair of Cys residues forms an intersubunit disulfide bond dependent on the redox conditions. CD spectroscopy revealed that the disulfide bond increases the thermal stability of the coiled-coil protein. We also reveal that two thiol modifier molecules are able to bind to Cys in a redox-dependent manner without disruption of the dimeric coiled-coil assembly. Thus, the biochemical properties of the cytoplasmic coiled-coil domain in the Hv channel depend on the redox condition, which may play a role in redox sensing in the phagosome.