The Biology Of Cyclic Nucleotides In E Coli

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（PEP）磷酸化，该磷酸化基团可以转移到第二组分（HPr）的活性位点。EI包含两个结构域，其活性受单体-二聚体平衡调节。新的研究表明，PEP和Mg这两种配体对二聚化有很强的促进作用，它们也能稳定和偶联两个结构域的展开；EI的磷酸化也促进二聚化。P-HPr可以与IIAglc相互作用并将一个磷酸基转移到IIAglc上。通过核磁共振分析确定了HPr-IIAglc配合物的溶液结构。HPr上的凸表面与IIAglc上的互补凹凹相互作用。两个结合表面都包含一个中心疏水核心区，周围环绕着一圈极性和带电残基，HPr为正，IIAglc为负。络合物的形成不涉及主链结构的改变，但涉及界面侧链的构象重排。虽然IIAglc的n端尾部不是HPr接受磷所必需的，但它是iicaglc给磷所必需的。结果表明，在存在磷脂的情况下，这条尾巴可以形成一个两亲螺旋，但在没有脂质的情况下，它是一个随机线圈。IIAglc还能调节乳糖渗透酶（LP）的活性。证实了IIAglc在底物促进下与LP的结合。LP突变影响结合；该研究表明，各种底物与LP结合会产生一系列独特的构象，每种构象都向膜的内表面呈现一个特定的表面，可以与IIAglc进行不同程度的相互作用。全局抑制因子Mlc与IICBglc的去磷酸形式结合，但不与磷酸形式结合。这种结合通过将Mlc从其靶序列中置换而导致Mlc调控基因的转录。因此，Mlc调控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，该酶直接招募Mlc来抑制其调控。共撰写了5篇综述文章或书籍章节，描述了涉及PTS蛋白的蛋白-蛋白复合物的三维结构或HPr对糖原磷酸化酶活性的调节。

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.