Activity-Based Probe for Histidine Kinase Signaling
Activity-Based Probe for Histidine Kinase Signaling
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DOI：
10.1021/ja3041702
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发表时间：
2012-06-06
影响因子：
15
通讯作者：
Carlson, Erin E.
中科院分区：
文献类型：
--
作者：
Wilke, Kaelyn E.;Francis, Samson;Carlson, Erin E.
细菌双组分系统（TCS）是由两种蛋白质组成的信号通路：组氨酸激酶（HK）和反应调节因子（RR）。在刺激下，HK在保守的组氨酸处自磷酸化。磷酰基随后转移到RR上的天冬氨酸，引发适应性反应，通常上调或下调基因表达。TCS信号控制细菌中的许多功能，包括发育，毒力和抗生素抗性，使这些系统中涉及的蛋白质成为潜在的治疗靶点。目前缺乏对HK进行分析的有效方法。为了直接读出HK活性，我们试图设计一种能够检测磷酸转移事件的探针;然而，由于P-N键的不稳定性，磷酸组氨酸种类的分析变得困难。我们预计，使用γ-硫代磷酸化ATP类似物，这将产生一个硫代磷酸化组氨酸中间体，可以克服这一挑战。我们确定，荧光团缀合的探针，BODIPY-FL-ATP γ S，标记活性HK蛋白，并竞争ATP结合位点。这种基于活性的探针为分析TCS和其他HK介导的过程提供了一种新的策略，并将有助于功能研究和抑制剂鉴定。
Bacterial two-component systems (TCSs) are signaling pathways composed of two proteins: a histidine kinase (HK) and a response regulator (RR). Upon stimulation, the HK autophosphorylates at a conserved histidine. The phosphoryl group is subsequently transferred to an aspartate on an RR, eliciting an adaptive response, often up- or downregulation of gene expression. TCS signaling controls many functions in bacteria, including development, virulence, and antibiotic resistance, making the proteins involved in these systems potential therapeutic targets. Efficient methods for the profiling of HKs are currently lacking. For direct readout of HK activity, we sought to design a probe that enables detection of the phosphotransfer event; however, analysis of the phosphohistidine species is made difficult by the instability of the P-N bond. We anticipated that use of a gamma-thiophosphorylated ATP analogue, which would yield a thiophosphorylated histidine intermediate, could overcome this challenge. We determined that the fluorophore-conjugated probe, BODIPY-FL-ATP gamma S, labels active HK proteins and is competitive for the ATP binding site. This activity-based probe provides a new strategy for analysis of TCSs and other HK-mediated processes and will facilitate both functional studies and inhibitor identification.