THE BIOLOGY OF CYCLIC NUCLEOTIDES IN E COLI

大肠杆菌中环核苷酸的生物学

• 批准号: 6290346
• 负责人: ALAN PETERKOFSKY
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290346
• 关键词: Escherichia coli; Mycoplasma; X ray crystallography; bacterial genetics; bacterial proteins; enzyme complex; enzyme mechanism; enzyme structure; gene expression; genetic transcription; intermolecular interaction; microorganism metabolism; phosphodiesterases; phosphorylation; phosphotransferases; protein structure; protein structure function; transport proteins

Structural and regulatory studies on protein components of the Escherichia coli (E. coli) sugar transport system known as the phosphoenolpyruvate: sugar phosphotransferase system (PTS) continued. The first component of the PTS (enzyme I, EI) is phosphorylated by phosphoenolpyruvate on an active site histidine and that phosphoryl group can be transferred to the active site of the second component (HPr). Previous studies have led to the elucidation of the three- dimensional structures of both the amino-terminal domain of EI (EIN) and HPr. The new studies were designed to describe, by NMR spectroscopy, the nature of the interface between EIN and HPr when the two proteins form a complex. The complex between EIN and HPr is a classical example of surface complementarity, involving an essentially all helical interface, comprising helices 2, 2, 3 and 4 of the alpha- subdomain of EIN and helices 1 and 2 of HPr, that requires virtually no changes in conformation of the components relative to that in their respective free states. The specificity of the complex is dependent on the correct placement of both van der Waals and electrostatic contacts. The transition state can be formed with minimal changes in overall conformation, and is stabilized in favor of phosphorylated HPr, thereby accounting for the directionality of phosphoryl transfer. EI undergoes a slow monomer-dimer transition. In vitro autophosphorylation of EI was studied at limiting concentrations of EI. Addition to incubation mixtures containing wild-type EI of inactive or low-activity mutant forms of EI resulted in stimulation of autophosphorylation activity. The kinetics of the activation fit well to a model in which the active form of EI is the dimer. These experiments provide support for the argument that only the dimeric form of EI can be autophosphorylated. One of the PTS proteins that can accept a phosphoryl group from P-HPr is named IIAglc. This protein also plays a role in the regulation of activity of other sugar transport systems in E. coli. By using a previously described direct binding assay, a collection of single-Cys replacement mutants in cytoplasmic loops of lactose permease was evaluated for their capacity to bind IIAglc. Selected Cys replacements in loops IV/V or VI/VII result in loss of binding activity. Analysis of the mutagenesis results together with multiple sequence alignments of a family of proteins that interacts with IIAglc provides the basis for developing two regions of consensus sequence in those partner proteins necessary for binding to IIAglc. The requirement for two interaction regions is interpreted in the regulatory framework of a substrate- dependent conformational change that brings these two regions into an orientation optimal for binding IIAglc. - E. coli, sugar transport, PTS, NMR, protein structure, EI dimerization, lactose permease-IIAglc complex

对大肠杆菌（E. coli）糖转运系统磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）的蛋白质组分进行了结构和调控研究。PTS的第一组分（酶I， EI）在活性位点组氨酸上被磷酸烯醇丙酮酸磷酸化，该磷酸化基团可以转移到第二组分（HPr）的活性位点。先前的研究已经阐明了EI （EIN）和HPr的氨基末端结构域的三维结构。新的研究旨在通过核磁共振光谱来描述当两种蛋白质形成复合物时EIN和HPr之间界面的性质。EIN和HPr之间的复合物是表面互补性的典型例子，涉及本质上所有的螺旋界面，包括EIN的α -子结构域的螺旋2、2、3和4和HPr的螺旋1和2，这实际上不需要相对于它们各自自由状态的组分的构象改变。配合物的特异性取决于范德华接触和静电接触的正确位置。过渡态可以在整体构象变化最小的情况下形成，并且有利于磷酸化HPr的稳定，从而解释了磷酰转移的方向性。EI经历了一个缓慢的单体-二聚体转变。在限定浓度的EI下，研究了EI的体外自磷酸化。在含有野生型EI的孵育混合物中加入无活性或低活性突变型EI，可刺激自磷酸化活性。活化动力学很好地符合EI的活性形式为二聚体的模型。这些实验支持了只有二聚体形式的EI才能被自磷酸化的观点。其中一种可以接受P-HPr磷酸化基的PTS蛋白被命名为IIAglc。该蛋白还在大肠杆菌中其他糖转运系统的活性调节中起作用。通过使用先前描述的直接结合试验，对乳糖渗透酶细胞质环中的单cys替代突变体的集合进行了评估，以评估其结合IIAglc的能力。在IV/V或VI/VII环中选择的Cys替换导致结合活性丧失。对诱变结果的分析以及与IIAglc相互作用的蛋白家族的多个序列比对，为在与IIAglc结合所需的伴侣蛋白中建立两个一致序列区域提供了基础。对两个相互作用区域的要求在一个底物依赖的构象变化的调节框架中得到解释，该变化使这两个区域进入一个最适合结合IIAglc的方向。-大肠杆菌，糖转运，PTS， NMR，蛋白质结构，EI二聚化，乳糖- iiaglc复合物