The Biology Of Cyclic Nucleotides In E Coli

大肠杆菌中环核苷酸的生物学

• 批准号: 6690446
• 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/6690446
• 关键词: Escherichia coli; Mycoplasma; X ray crystallography; acetylation; active sites; bacterial genetics; bacterial proteins; carbohydrate transport; enzyme complex; enzyme mechanism; enzyme structure; gene expression; genetic transcription; lactose; microorganism metabolism; nuclear magnetic resonance spectroscopy; permease; phosphodiesterases; phosphorylation; phosphotransferases; protein engineering; protein protein interaction; protein structure; protein structure function

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 (PEP) 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 and its activity is regulated by a monomer-dimer equilibrium. New studies demonstrated that dimerization is strongly promoted by PEP and Mg, ligands which also stabilize and couple the unfolding of the two domains; phosphorylation of EI also promotes dimerization. P-HPr can interact with and transfer a phosphoryl group to IIAglc. The solution structure of the HPr-IIAglc complex was solved by NMR. A convex surface on HPr interacts with a complementary concave depression on IIAglc. Both binding surfaces comprise a central hydrophobic core region surrounded by a ring of polar and charged residues, positive for HPr and negative for IIAglc. Complex formation involves no change in backbone structures, but some conformational rearrangements of interfacial sidechains. While the N-terminal tail of IIAglc is not required for phosphoacceptance from HPr, it is necessary for phosphodonation to IICBglc. It was demonstrated that this tail can form an amphipathic helix in the presence of phospholipids, but is a random coil in the absence of lipid. IIAglc also regulates the activity of lactose permease (LP). Binding of IIAglc, promoted by substrate, to LP was demonstrated. Mutations in LP affect the binding; the study suggested that binding of various substrates to LP results in a collection of unique conformations, each of which presents a specific surface toward the inner face of the membrane that can interact to varying degrees with IIAglc. The global repressor, Mlc, binds to the dephospho-, but not the phospho-form of IICBglc. The binding results in transcription of Mlc-regulated genes by displacing Mlc from its target sequences. Therefore, the glucose induction of Mlc-regulated genes is caused by dephosphorylation of the membrane-bound transporter enzyme IICBglc, which directly recruits Mlc to derepress its regulon. A total of five review articles or book chapters were written describing the three dimensional structures of protein-protein complexes involving PTS proteins or the regulation by HPr of glycogen phosphorylase activity.

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（PEP）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含有两个结构域，其活性受单体-二聚体平衡调节。新的研究表明，PEP和Mg强烈促进二聚化，这两种配体也稳定和偶联两个结构域的解折叠; EI的磷酸化也促进二聚化。P-HPr可与IIAglc相互作用并将磷酰基转移至IIAglc。HPr-IIAglc配合物的溶液结构通过NMR解析。HPr上的凸面与IIAglc上的互补凹面相互作用。两个结合表面都包含被极性和带电残基环包围的中心疏水核心区域，其对HPr为阳性，对IIAglc为阴性。复合物的形成不涉及主链结构的变化，但界面侧链的一些构象重排。虽然IIAglc的N-末端尾对于HPr的磷酸接受不是必需的，但对于IICBglc的磷酸供体是必需的。结果表明，该尾在磷脂存在下可以形成两亲性螺旋，但在不存在脂质的情况下是无规卷曲。IIAglc还调节乳糖通透酶（LP）的活性。证明了底物促进IIAglc与LP的结合。LP中的突变影响结合;该研究表明，各种底物与LP的结合导致了一系列独特的构象，其中每一种构象都呈现出朝向膜内表面的特定表面，这些表面可以不同程度地与IIAglc相互作用。全局阻遏物Mlc与IICBglc的去磷酸化形式结合，但不与磷酸化形式结合。这种结合通过从其靶序列置换Mlc而导致Mlc调节的基因的转录。因此，Mlc调节基因的葡萄糖诱导是由膜结合转运酶IICBglc的去磷酸化引起的，其直接募集Mlc以去抑制其调节子。总共有五篇综述文章或书籍章节描述了涉及PTS蛋白的蛋白质-蛋白质复合物的三维结构或HPr对糖原磷酸化酶活性的调节。