Ben-Zeev Efrat, Fux Liat, Amster-Choder Orna, Eisenstein Miriam
Department of Chemical Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
J Mol Biol. 2005 Apr 8;347(4):693-706. doi: 10.1016/j.jmb.2005.01.068.
BglG and LicT are transcriptional antiterminators from Escherichia coli and Bacillus subtilis, respectively, that control the expression of genes and operons involved in transport and catabolism of carbohydrates. Both proteins contain a duplicate conserved domain, the PTS-regulation domain (PRD), and they are regulated by phosphorylation on specific, highly conserved histidine residues located in the PRDs. However, despite their similar function and the high sequence identity, experimental evidence implies different modes of regulation. Thus, BglG must be de-phosphorylated on PRD2 in order to form active dimers, whereas activation of LicT requires de-phosphorylation on PRD1 and phosphorylation on PRD2. Here we address two goals. First, we test in vivo and in silico the effect of point mutations in the PRDs of BglG on the PRD-PRD dimerization. Second, we explore computationally the effect of histidine phosphorylation on PRD dimerization in BglG and LicT. We find excellent correspondence between the experimental and computational measures of the influence of mutations on PRD dimerization in BglG. This establishes that the geometric-electrostatic complementarity scores computed with the program MolFit provide a good measure of the effects of mutations in this system. In addition, it indicates that the dimerization mode of the separately expressed PRDs of BglG is similar to the dimers formed by activated LicT. The computations also show that phosphorylation of the histidine residues in PRD1 of either BglG or LicT leads to a strong electrostatic repulsion. Conversely, the phosphorylation of one histidine residue in PRD2 of LicT leads to improved electrostatic complementarity at the PRD2-PRD2 interface, whereas the corresponding phosphorylation in BglG has negligible contribution. This different conduct may be attributed to a single replacement in the sequence of PRD2 in BglG compared to LicT, Ala262 versus Asp261, respectively.
BglG和LicT分别是来自大肠杆菌和枯草芽孢杆菌的转录抗终止因子，它们控制参与碳水化合物运输和分解代谢的基因及操纵子的表达。这两种蛋白质都含有一个重复的保守结构域，即磷酸转移酶系统调节结构域（PRD），并且它们通过位于PRD中的特定、高度保守的组氨酸残基上的磷酸化作用进行调节。然而，尽管它们功能相似且序列同一性高，但实验证据表明它们的调节模式不同。因此，BglG必须在PRD2上脱磷酸化才能形成活性二聚体，而LicT的激活则需要PRD1脱磷酸化和PRD2磷酸化。在此我们有两个目标。第一，我们在体内和计算机模拟中测试BglG的PRD中位点突变对PRD - PRD二聚化的影响。第二，我们通过计算探索组氨酸磷酸化对BglG和LicT中PRD二聚化的影响。我们发现关于突变对BglG中PRD二聚化影响的实验测量和计算测量之间具有极好的一致性。这表明用MolFit程序计算的几何 - 静电互补性得分能很好地衡量该系统中突变的影响。此外，这表明BglG单独表达的PRD的二聚化模式与激活的LicT形成的二聚体相似。计算还表明，BglG或LicT的PRD1中组氨酸残基的磷酸化会导致强烈的静电排斥。相反，LicT的PRD2中一个组氨酸残基的磷酸化会导致PRD2 - PRD2界面处静电互补性提高，而BglG中相应的磷酸化作用贡献可忽略不计。这种不同的行为可能归因于与LicT相比，BglG中PRD2序列的一个单一替换，分别为Ala262与Asp261。
