Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, New Jersey, United States of America.
PLoS Biol. 2011 Feb 8;9(2):e1000589. doi: 10.1371/journal.pbio.1000589.
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 家族蛋白在枯草芽孢杆菌中得到了最为广泛的研究，它们调节的活性包括孢子形成、遗传能力、抗生素表达和 ICEBs1 转座子的运动。Rap 蛋白的一个亚类由磷酸酶组成，通过去磷酸化反应调节子 Spo0F，从而控制枯草芽孢杆菌和炭疽芽孢杆菌的孢子形成。Rap 磷酸酶活性的机制尚不清楚。在这里，我们呈现了 RapH-Spo0F 的 X 射线晶体结构，该结构显示 Rap 蛋白由 3 个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 RapH 3 螺旋束中谷氨酰胺的催化作用，该谷氨酰胺插入 Spo0F 的活性位点。我们表明，RapH 不仅可以通过空间位阻阻止磷酸基团从 Spo0F 转移到 Spo0F 上来拮抗孢子形成，还可以去磷酸化 Spo0F。我们对 RapH-Spo0F 相互作用的结构-功能分析确定了 Rap 蛋白中对 Spo0F 磷酸酶活性至关重要的残基。这些信息使我们能够仅根据序列将 Rap 蛋白的 Spo0F 磷酸酶活性分配给一个 Rap 蛋白，这在以前是不可能的。最后，作为我们对 Rap 磷酸酶功能的结构要求的新发现的理解的最终检验，我们通过理性设计一个抑制应答调节蛋白 ComA 与 DNA 结合的非磷酸酶 Rap 蛋白，使其能够去磷酸化 Spo0F。除了揭示 Rap 蛋白对应答调节蛋白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节的 T 环-Y 变构模型，该模型限制了芳香“开关”残基在β4-α4 环采用接近活性位点的构象时处于内部位置。
