Nitric Oxide-Induced Conformational Changes Govern H-NOX and Histidine Kinase Interaction and Regulation in Shewanella oneidensis.
Nitric Oxide-Induced Conformational Changes Govern H-NOX and Histidine Kinase Interaction and Regulation in Shewanella oneidensis.
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DOI：
10.1021/acs.biochem.6b01133
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发表时间：
2017-02
期刊：
影响因子：
2.9
通讯作者：
Minxi Rao;M. Herzik;A. Iavarone;M. Marletta
中科院分区：
文献类型：
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作者：
Minxi Rao;M. Herzik;A. Iavarone;M. Marletta
一氧化氮 (NO) 通过血红素一氧化氮/氧结合 (H-NOX) 蛋白信号传导参与多个细菌家族的生物膜调节。 Shewanella oneidensis H-NOX (So H-NOX) 与同一操纵子上编码的组氨酸激酶 (So HnoK) 相关，它们一起形成多组分信号网络，So H-NOX 的 NO 结合状态抑制 So HnoK 自磷酸化活性，影响三个反应调节因子的磷酸化状态。尽管 So H-NOX 在 NO 结合后的构象变化已得到结构表征，但 NO 结合的 So H-NOX 抑制 HnoK 的机制仍不清楚。在本研究中，表征了 So H-NOX 和 So HnoK 相互作用和调节的分子细节。 So HnoK 中的 N 端结构域被确定为 H-NOX 相互作用的位点，并结合氢-氘交换质谱和表面扫描诱变鉴定了 So H-NOX 上的结合界面。结合动力学测量和分析凝胶过滤表明，与未配体状态的 H-NOX 相比，NO 结合的 So H-NOX 对 So HnoK 具有更紧密的亲和力，将结合亲和力与激酶抑制相关联。结合缺陷的 H-NOX 突变体的激酶活性测定进一步表明，虽然 H-NOX-HnoK 复合物的形成是 HnoK 催化活性所必需的，但 NO 结合时的 H-NOX 构象变化是 HnoK 抑制所必需的。
Nitric oxide (NO) is implicated in biofilm regulation in several bacterial families via heme-nitric oxide/oxygen binding (H-NOX) protein signaling. Shewanella oneidensis H-NOX (So H-NOX) is associated with a histidine kinase (So HnoK) encoded on the same operon, and together they form a multicomponent signaling network whereby the NO-bound state of So H-NOX inhibits So HnoK autophosphorylation activity, affecting the phosphorylation state of three response regulators. Although the conformational changes of So H-NOX upon NO binding have been structurally characterized, the mechanism of HnoK inhibition by NO-bound So H-NOX remains unclear. In the present study, the molecular details of So H-NOX and So HnoK interaction and regulation are characterized. The N-terminal domain in So HnoK was determined to be the site of H-NOX interaction, and the binding interface on So H-NOX was identified using a combination of hydrogen-deuterium exchange mass spectrometry and surface-scanning mutagenesis. Binding kinetics measurements and analytical gel filtration revealed that NO-bound So H-NOX has a tighter affinity for So HnoK compared that of H-NOX in the unliganded state, correlating binding affinity with kinase inhibition. Kinase activity assays with binding-deficient H-NOX mutants further indicate that while formation of the H-NOX-HnoK complex is required for HnoK to be catalytically active, H-NOX conformational changes upon NO-binding are necessary for HnoK inhibition.