The Biology Of Sugar Transport in E Coli

继续对大肠杆菌糖转运系统磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）的蛋白质组分进行结构和调控研究。与Clore实验室（NIDDK）合作的新研究已经通过核磁共振阐明了48kda的IIAMannose-HPr复合物的三维结构。甘露糖是二聚体，每个二聚体对HPr有两个对称相关的结合位点。HPr上的凸表面与iamannose的两个亚基界面处的深凹槽相互作用。iamannose上的相互作用表面主要是螺旋形的。两种蛋白的结合位点在疏水、亲水性和带电残基的形状和分布上是互补的。iamannose和HPr的活性位点组氨酸非常接近。

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.