Structural basis of response regulator dephosphorylation by Rap phosphatases.
Structural basis of response regulator dephosphorylation by Rap phosphatases.
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DOI：
10.1371/journal.pbio.1000589
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发表时间：
2011-02-08
期刊：
影响因子：
9.8
通讯作者：
Neiditch MB
中科院分区：
文献类型：
--
作者：
Parashar V;Mirouze N;Dubnau DA;Neiditch MB
晶体学、生化和遗传学研究揭示了芽孢杆菌中Rap蛋白磷酸酶活性在磷传递途径中导致孢子形成的机制。细菌Rap家族蛋白在枯草芽孢杆菌中得到了最广泛的研究，它们调节包括产孢、遗传能力、抗生素表达和ICEBs1转座子的运动在内的活动。Rap蛋白的一个子集由磷酸酶组成，磷酸酶通过使反应调节因子Spo0F去磷酸化来控制枯草芽孢杆菌和炭疽芽孢杆菌的产孢。Rap磷酸酶活性的机制基础尚不清楚。RapH-Spo0F x射线晶体结构表明，Rap蛋白由一个3-螺旋束和一个四肽重复结构域组成。广泛的生化和遗传功能研究揭示了观察到的RapH-Spo0F相互作用的重要性，包括谷氨酰胺在插入Spo0F活性位点的RapH 3-螺旋束中的催化作用。我们发现，除了使Spo0F去磷酸化外，RapH还可以通过立体阻断磷酸基与Spo0F之间的转移来拮抗孢子形成。我们对RapH-Spo0F相互作用的结构-功能分析发现Rap蛋白残基对Spo0F磷酸酶活性至关重要。这一信息使我们能够仅根据序列将Spo0F磷酸酶活性分配给Rap蛋白，这在以前是不可能的。最后，作为我们对Rap磷酸酶功能结构要求的新认识的最终测试，一种抑制反应调节因子ComA与DNA结合的非磷酸酶Rap蛋白被合理地设计为去磷酸化Spo0F。除了揭示Rap蛋白应答调节因子去磷酸化的机制基础外，我们的研究还支持了先前提出的受体结构域调控的t -环- y变构模型，即当β4-α4环采用活性位点近端构象时，芳香“开关”残基限制在内部位置。磷接力信号转导途径调节多种芽孢杆菌的产孢，包括遗传模式生物枯草芽孢杆菌和炭疽芽孢杆菌。组氨酸激酶启动磷酸化基团沿磷接力途径的流动，然后将它们运送到下游的反应调节转录因子Spo0A。最终，孢子形成是由磷酸化的Spo0A的细胞浓度控制的。在许多芽孢杆菌中，Rap磷酸酶的作用与组氨酸激酶相反，通过使一种称为Spo0F的中间途径蛋白去磷酸化来抑制Spo0A的激活。在这里，我们展示了Rap蛋白RapH与Spo0F复合物的结构，这是通过x射线晶体学确定的。RapH-Spo0F结构以及生化和遗传学研究揭示了rapp蛋白介导的Spo0F去磷酸化机制。我们利用从结构-功能分析中收集到的信息，首先，根据序列将Spo0F磷酸酶活性分配给未表征的Rap蛋白RapJ，其次，将Spo0F磷酸酶活性重新设计为非磷酸酶Rap蛋白RapF。我们发现除了使Spo0F去磷酸化外，Rap蛋白还可以通过立体阻断磷酸基与Spo0F之间的转移来抑制孢子形成磷酸化接力。最终，可能会开发出新型药物，通过模仿Rap蛋白对反应调节因子的拮抗作用，破坏磷酸化基团沿磷转移信号通路的流动。
Crystallographic, biochemical, and genetic studies reveal the mechanism of Rap protein phosphatase activity within the phosphorelay pathway leading to sporulation in Bacillus species. Bacterial Rap family proteins have been most extensively studied in Bacillus subtilis, where they regulate activities including sporulation, genetic competence, antibiotic expression, and the movement of the ICEBs1 transposon. One subset of Rap proteins consists of phosphatases that control B. subtilis and B. anthracis sporulation by dephosphorylating the response regulator Spo0F. The mechanistic basis of Rap phosphatase activity was unknown. Here we present the RapH-Spo0F X-ray crystal structure, which shows that Rap proteins consist of a 3-helix bundle and a tetratricopeptide repeat domain. Extensive biochemical and genetic functional studies reveal the importance of the observed RapH-Spo0F interactions, including the catalytic role of a glutamine in the RapH 3-helix bundle that inserts into the Spo0F active site. We show that in addition to dephosphorylating Spo0F, RapH can antagonize sporulation by sterically blocking phosphoryl transfer to and from Spo0F. Our structure-function analysis of the RapH-Spo0F interaction identified Rap protein residues critical for Spo0F phosphatase activity. This information enabled us to assign Spo0F phosphatase activity to a Rap protein based on sequence alone, which was not previously possible. Finally, as the ultimate test of our newfound understanding of the structural requirements for Rap phosphatase function, a non-phosphatase Rap protein that inhibits the binding of the response regulator ComA to DNA was rationally engineered to dephosphorylate Spo0F. In addition to revealing the mechanistic basis of response regulator dephosphorylation by Rap proteins, our studies support the previously proposed T-loop-Y allostery model of receiver domain regulation that restricts the aromatic “switch” residue to an internal position when the β4-α4 loop adopts an active-site proximal conformation. A phosphorelay signal transduction pathway regulates sporulation in numerous Bacillus species including the genetic model organism, B. subtilis, and the causative agent of anthrax, B. anthracis. Histidine kinases initiate the flow of phosphoryl groups along the phosphorelay pathway, which then shuttles them to a downstream response-regulator transcription factor called Spo0A. Ultimately, sporulation is governed by the cellular concentration of phosphorylated Spo0A. In numerous Bacillus species, Rap phosphatases function in opposition to the histidine kinases, inhibiting Spo0A activation by dephosphorylating an intermediate pathway protein called Spo0F. Here we present the structure of a Rap protein, RapH, in complex with Spo0F, as determined by X-ray crystallography. The RapH–Spo0F structure, along with biochemical and genetic studies, reveals the mechanism of Rap-protein-mediated Spo0F dephosphorylation. We used information gleaned from our structure–function analysis, first, to assign Spo0F phosphatase activity to an uncharacterized Rap protein, RapJ, on the basis of sequence alone, and, second, to engineer Spo0F phosphatase activity de novo into a non-phosphatase Rap protein, RapF. We found that in addition to dephosphorylating Spo0F, Rap proteins can inhibit the sporulation phosphorelay by sterically blocking the transfer of phosphoryl groups to and from Spo0F. Ultimately, new classes of drugs might be developed that disrupt the flow of phosphoryl groups along phosphotransfer signaling pathways by mimicking the antagonistic effects of Rap proteins on response regulators.
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