THE BIOLOGY OF CYCLIC NUCLEOTIDES IN E COLI

对大肠杆菌糖运输系统--磷酸烯醇式丙酮酸：糖磷酸转移酶系统(PTS)的蛋白质组分的结构和调控的研究仍在继续。PTS的第一组分(酶I，EI)被活性部位组氨酸上的磷酸烯醇式丙酮酸磷酸化，该磷酸化基团可以转移到第二组分的活性部位(HPR)。以往的研究已经阐明了EI(EIN)和HPR的氨基末端结构域的三维结构。这项新的研究旨在通过核磁共振波谱来描述当两种蛋白质形成复合体时，EIN和HPR之间的界面的性质。EIN和HPR之间的络合物是表面互补的经典例子，涉及基本上全螺旋界面，包括EIN的α亚区的螺旋2、2、3和4以及HPR的螺旋1和2，这几乎不需要改变组分相对于其各自自由态的构象。该复合体的特异性取决于范德华触点和静电触点的正确位置。过渡态可以在总构象变化很小的情况下形成，并有利于磷酸化HPR的稳定，从而解释了磷酸基转移的方向性。EI经历了缓慢的单体-二聚体转变。在体外研究了EI在限定浓度下的自磷酸化。除了含有野生型EI的非活性或低活性突变形式的EI的孵育混合物外，还能刺激自磷酸化活性。活化动力学符合EI的活性形式为二聚体的模型。这些实验支持了只有EI的二聚体形式才能自动磷酸化的论点。一种能够接受P-HPR的磷酰基的PTS蛋白被命名为IIAglc。该蛋白还在调节大肠杆菌中其他糖运输系统的活性方面发挥作用。通过使用先前描述的直接结合试验，对乳糖渗透酶胞质环中的一组单半胱氨酸替换突变体与IIAglc的结合能力进行了评估。在IV/V或VI/VII环中选择的Cys替换会导致结合活性的丧失。对突变结果的分析以及与IIAglc相互作用的一系列蛋白质的多个序列比对，为在那些与IIAglc结合所需的伙伴蛋白中开发两个共同序列区域提供了基础。对两个相互作用区的要求在底物依赖的构象变化的调控框架中被解释，该变化使这两个区域进入适合结合IIAglc的方向。-大肠杆菌，糖转运，PTS，核磁共振，蛋白质结构，EI二聚，乳糖渗透酶-IIAglc复合体

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