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The Biology Of Sugar Transport in E Coli

大肠杆菌中糖运输的生物学

基本信息

批准号：
7154184
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
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

Structural and regulatory studies on protein components of the E. coli sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (PTS) continued. New studies, in collaboration with the Clore laboratory (NIDDK), have elucidated the three-dimensional structure, by NMR, of the 48-kDa IIAMannose-HPr complex. IIAMannose is dimeric and has two symmetrically related binding sites per dimer for HPr. A convex surface on HPr interacts with a deep groove at the interface of the two subunits of IIAMannose. The interaction surface on IIAMannose is predominantly helical. The binding sites on the two proteins are complementary in terms of shape and distribution of hydrophobic, hydrophilic and charged residues. The active site histidines of IIAMannose and HPr are in close proximity. We have also been carrying out structural and functional studies on a pathway, paralagous to the sugar transport system, known as the nitrogen regulatory PTS. The protein currently under study is the paralog of the sugar transporter IIAGlucose, referred to as IIANitrogen. In collaboration with the Wang laboratory (Omaha, Nebraska), we have elucidated, by NMR, the solution structure of IIANitrogen as well as its interaction with its partner protein NPr, a paralog of HPr of the sugar transport system. The diffusion coefficient indicates that the functional form of IIANitrogen is a monomer (~18 kDa) in solution. Thus, the dimeric structure of the protein found in the crystal is an artifact of crystal packing. The residual dipolar coupling data are consistent with the structure in solution matching that of molecule A of the crystal structure. Chemical shift mapping identified the surface on IIANitrogen for NPr binding. In collaboration with the Seok laboratory (Seoul, Korea), we have investigated the biological function of IIANitrogen. We carried out phenotype microarray analysis on a mutant deleted for the gene expressing the first enzyme of the nitrogen regulatory PTS (Enzyme INitrogen). The findings of these studies, suggesting resistance to the growth inhibitory effects of certain peptides, led to growth studies with a mutant deleted for the gene encoding IIANitrogen. These studies revealed that the IIANitrogen mutant was extremely sensistive to leucine-containing peptides. The toxicity of leucine-containing peptides was found to be due to leucine and the dephospho-form of IIANitrogen was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho-form of IIANitrogen is required for derepression of the ilvBN operon encoding acetohydroxyacid synthetase, which catalyzes the first step common to the biosynthesis of the branched-chain amino acids.

对大肠杆菌的蛋白质组分进行了结构和调控研究。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）继续。与Clore实验室（NIDDK）合作的新研究通过NMR阐明了48 kDa IIA甘露糖-HPr复合物的三维结构。IIA甘露糖是二聚体，每个二聚体有两个对称相关的HPr结合位点。HPr上的凸表面与IIA甘露糖的两个亚基的界面处的深沟相互作用。IIA甘露糖上的相互作用表面主要是螺旋形的。两种蛋白质上的结合位点在疏水、亲水和带电残基的形状和分布方面是互补的。IIAMannose和HPr的活性位点组氨酸非常接近。我们还一直在进行结构和功能研究的途径，糖转运系统，称为氮调节PTS。目前正在研究的蛋白质是糖转运蛋白IIAG葡萄糖的蛋白质，称为IIANitrogen。在与王实验室（奥马哈，内布拉斯加州）合作，我们已经阐明，通过NMR，溶液结构的IIANitrogen以及它的相互作用与它的伙伴蛋白质NPr，一个paramount的HPr的糖运输系统。扩散系数表明，IIAN氮的功能形式是溶液中的单体（~18 kDa）。因此，在晶体中发现的蛋白质的二聚体结构是晶体堆积的人工产物。残余偶极耦合数据与溶液中的结构一致，与晶体结构的分子A的结构相匹配。化学位移图确定了NPr结合的IIANitrogen表面。与Seok实验室（韩国首尔）合作，我们研究了IIANitrogen的生物学功能。我们对缺失表达氮调节PTS的第一种酶（Enzyme INitrogen）的基因的突变体进行了表型微阵列分析。这些研究的结果表明，对某些肽的生长抑制作用具有抗性，导致了对缺失编码IIAN氮的基因的突变体的生长研究。这些研究表明，IIAN氮突变体对含亮氨酸的肽非常敏感。发现含亮氨酸的肽的毒性是由于亮氨酸，并且发现脱磷酸形式的IIAN氮是中和亮氨酸毒性所必需的。进一步的研究表明，IIAN氮的脱磷酸形式是编码乙酰羟酸合成酶的ilvBN操纵子的去阻遏所必需的，所述乙酰羟酸合成酶催化支链氨基酸的生物合成所共有的第一步。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Solution structure of the N-terminal amphitropic domain of Escherichia coli glucose-specific enzyme IIA in membrane-mimetic micelles.

膜模拟胶束中大肠杆菌葡萄糖特异性酶 IIA 的 N 端两亲性结构域的溶液结构。

DOI：
10.1110/ps.0301503
发表时间：
2003
期刊：
Protein science : a publication of the Protein Society
影响因子：
0
作者：
Wang,Guangshun;Keifer,PaulA;Peterkofsky,Alan
通讯作者：
Peterkofsky,Alan

A novel membrane anchor function for the N-terminal amphipathic sequence of the signal-transducing protein IIAGlucose of the Escherichia coli phosphotransferase system.

大肠杆菌磷酸转移酶系统信号转导蛋白 IIAGlucose N 端两亲序列的新型膜锚定功能。

DOI：
10.1074/jbc.c000709200
发表时间：
2000
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Wang,G;Peterkofsky,A;Clore,GM
通讯作者：
Clore,GM

Deduction of consensus binding sequences on proteins that bind IIAGlc of the phosphoenolpyruvate:sugar phosphotransferase system by cysteine scanning mutagenesis of Escherichia coli lactose permease.

通过大肠杆菌乳糖通透酶的半胱氨酸扫描诱变推导结合磷酸烯醇丙酮酸：糖磷酸转移酶系统的 IIAGlc 的蛋白质上的共有结合序列。

DOI：
10.1073/pnas.96.7.3525
发表时间：
1999
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Sondej,M;Sun,J;Seok,YJ;Kaback,HR;Peterkofsky,A
通讯作者：
Peterkofsky,A

Topography of the surface of the Escherichia coli phosphotransferase system protein enzyme IIAglc that interacts with lactose permease.

与乳糖通透酶相互作用的大肠杆菌磷酸转移酶系统蛋白 IIAglc 的表面形貌。