The Biology Of Sugar Transport in E Coli

对大肠杆菌糖运输系统的蛋白质组分的结构和调控的研究仍在继续。与Clore实验室(NIDDK)合作的新研究通过核磁共振阐明了48 kDa IIA甘露-HPR复合体的三维结构。IIA甘露糖是二聚体，每个二聚体与HPR有两个对称相关的结合位点。HPR上的凸面与IIA甘露糖两个亚单位界面上的深凹槽相互作用。IIA型甘露糖上的相互作用面主要是螺旋的。这两种蛋白质的结合部位在疏水残基、亲水残基和带电残基的形状和分布上是互补的。IIAMANNSE和HPR的活性部位组氨酸非常接近。 我们还一直在进行与糖运输系统平行的一条途径的结构和功能研究，称为氮调节PTS。目前正在研究的蛋白质是糖转运蛋白IIAG的Paralog，被称为IIANN。在与Wang实验室(内布拉斯加州奥马哈)的合作下，我们通过核磁共振阐明了IIANN的溶液结构以及它与其伙伴蛋白NPR的相互作用，NPR是糖运输系统的HPR的平行序列。扩散系数表明亚硝酸根在溶液中的官能化形式为单体(~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产物。剩余偶极耦合数据与溶液中的结构一致，与晶体结构中的A分子相匹配。化学位移图谱确定了IIANN上NPR结合的表面。 我们与Seok实验室(韩国首尔)合作，研究了IIANN的生物学功能。我们对一个表达氮素调节酶PTS第一酶基因缺失的突变体进行了表型芯片分析。这些研究的结果表明，对某些多肽的生长抑制作用具有抵抗力，导致生长研究中删除了编码IIANN基因的突变。这些研究表明，IIANN突变体对含有亮氨酸的多肽非常敏感。含有亮氨酸的多肽的毒性是由亮氨酸引起的，而IIANN的脱磷形式被发现是中和亮氨酸毒性所必需的。进一步的研究表明，IIANN的脱磷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，该操纵子催化了支链氨基酸生物合成的第一步。

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.