Structural Basis of Response Regulator Dephosphorylation by Rap Phosphatases

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.细菌Rap家族蛋白（Bacterial Rap family proteins）的研究最早且最为深入的场景是枯草芽孢杆菌（Bacillus subtilis），该类蛋白可调控多项生命活动，包括孢子形成、遗传感受态、抗生素表达以及ICEBs1转座子的转座行为。其中一类Rap蛋白属于磷酸酶家族，通过对应答调节因子Spo0F进行去磷酸化修饰，调控枯草芽孢杆菌和炭疽芽孢杆菌（Bacillus anthracis，简称B. anthracis）的孢子形成过程。此前，Rap磷酸酶活性的分子机制一直未被阐明。本研究解析了RapH与Spo0F复合物的X射线晶体结构，结果显示Rap蛋白由3螺旋束（3-helix bundle）和四肽重复结构域（tetratricopeptide repeat domain）构成。后续开展的大量生化与遗传功能实验证实，解析得到的RapH-Spo0F相互作用模式具有关键生物学意义，其中RapH 3螺旋束中的谷氨酰胺残基可插入Spo0F的活性位点，发挥催化功能。研究还发现，RapH除了可对Spo0F进行去磷酸化修饰外，还能通过空间位阻效应阻断Spo0F的磷酸基团转移过程，从而拮抗孢子形成通路。本次针对RapH-Spo0F相互作用的结构-功能分析，筛选得到了对Spo0F磷酸酶活性至关重要的Rap蛋白氨基酸残基。基于该研究结果，我们首次实现了仅通过蛋白序列即可预测Rap蛋白是否具有Spo0F磷酸酶活性的能力，而此前这一工作无法完成。最后，为验证我们对Rap磷酸酶功能结构基础的新认知，研究人员通过理性设计改造了一种原本不具备磷酸酶活性的Rap蛋白——该蛋白可通过抑制应答调节因子ComA与DNA的结合——使其获得了对Spo0F的去磷酸化活性。本研究不仅阐明了Rap蛋白介导应答调节因子去磷酸化的分子机制，同时也验证了此前提出的受体结构域调控的T-loop-Y变构模型：当β4-α4环处于活性位点附近构象时，该模型会将芳香族“开关”残基限制在内部位置。