THE BIOLOGY OF CYCLIC NUCLEOTIDES IN E COLI

大肠杆菌中环核苷酸的生物学

• 批准号: 6432612
• 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/6432612
• 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). EI contains two domains. The isolated recombinant helical domain was shown to bind to HPr. In vitro reconstitution experiments with isolated recombinant helical domain and a construct deficient in that domain showed that it was possible to recover autophosphorylation by PEP. Phosphotransfer activity was also recovered in reconstitution of the aminoterminal and carboxyterminal domains. Mutations at the active site of EI were engineered and studied by biophysical methods for determination of conformational stability. The order of conformational stability is His>Ala>Glu>His-PO3. The binding of HPr to active-site mutants is temperature-independent with about the same affinity constant. 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 region on the surface of IIAglc that interfaces with lactose permease was characterized. Actetylation of lysine residues by sulfosuccinimidyl acetate treatment of IIAglc reduced the degree of interaction with lactose permease. To localize the lysine residues on IIAglc that are involved in the regulatory interaction, selected lysine residues were mutagenized. Conversion of nine separate lysines to glutamic acid resulted in proteins that were still capable of phosphoryl acceptance from HPr. Except for Lys69, all the modified proteins were able to bind to lactose permease. A model for the region of the surface of IIAglc, including Lys69, that interacts with lactose permease was proposed. HPr is a multifunctional protein; as a phosphocarrier in the PTS, it interacts with both EI and IIAglc. In addition it is an allosteric regulator of glycogen phosphorylase. Because the nature of the surface of HPr that interacts with this multiplicity of proteins was previously undefined, those interactions were investigated by NMR. The chemical shift changes of the backbone and side chain amide 1H and 15N nuclei of uniformly 15N-labeled HPr in the presence and absence of natural abundance glycogen phosphorylase, IIAglc or the aminoterminal domain of EI were determined. Mapping those chemical shift perturbations on the three-dimensional structure of HPr permitted the identification of the binding surface on HPr for interaction with those proteins. The mapped interfaces are remarkably similar, indicating that HPr employs a similar syurface in binding to its partners.

对大肠杆菌（Escherichia coli，E.大肠杆菌）糖转运系统称为磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）。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，并且该磷酰基可以转移到第二组分（HPr）的活性位点。EI包含两个域。分离的重组螺旋结构域显示与HPr结合。用分离的重组螺旋结构域和该结构域缺陷的构建体进行的体外重建实验表明，PEP可以恢复自磷酸化。在氨基末端和羧基末端结构域的重建中也恢复了磷酸转移活性。在EI的活性位点处的突变被工程化，并通过生物物理方法进行研究以确定构象稳定性。构象稳定性顺序为His>Ala>Glu> His-PO 3。HPr与活性位点突变体的结合不依赖于温度，具有大约相同的亲和力常数。其中一种可以接受P-HPr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转运系统的活性中发挥作用。杆菌通过使用先前描述的直接结合测定，IIAglc表面上与乳糖通透酶界面的区域被表征。乙酰化的赖氨酸残基的磺基琥珀酰亚胺乙酸酯处理IIAglc降低了与乳糖通透酶的相互作用的程度。为了定位IIAglc上参与调控相互作用的赖氨酸残基，对选定的赖氨酸残基进行诱变。将9个独立的赖氨酸转化为谷氨酸产生的蛋白质仍然能够从HPr接受磷酰基。除Lys 69外，所有修饰蛋白均能与乳糖通透酶结合。IIAglc，包括Lys 69，与乳糖通透酶相互作用的表面区域的模型提出。HPr是一种多功能蛋白质;作为PTS中的磷酸载体，它与EI和IIAglc相互作用。此外，它还是糖原磷酸化酶的变构调节剂。由于HPr表面与多种蛋白质相互作用的性质以前未定义，因此通过NMR研究了这些相互作用。 化学位移的变化的骨干和侧链酰胺1H和15 N核的均匀15 N-标记HPr在存在和不存在的天然丰度糖原磷酸化酶，IIAglc或氨基末端结构域EI进行了测定。映射HPr的三维结构上的化学位移扰动允许识别HPr上的结合表面与这些蛋白质的相互作用。映射的接口非常相似，这表明HPr在绑定到其合作伙伴时使用了类似的syurface。