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。PTS的第一组分(酶I，EI)被活性部位组氨酸上的磷酸烯醇式丙酮酸(PEP)磷酸化，在需要镁(2+)的反应中生成丙酮酸。与Ann Ginsburg(见参考文献3)合作的新研究利用了Ei(H189A)的非活性突变体，其中丙氨酸取代了活性位点His189，在His189中，底物结合效应可以与磷酸化作用分开。1 mM的PEP强烈促进EI(H189A)的二聚化，5 mM的丙酮酸则相反。当丙酮酸抑制PEP产生的N-端结构域和C-端结构域之间的偶联时，EI(H189A)的二聚化常数从&gt；10的8次方降至&lt；5×10的5次方。PEP与EI(H189A)的一个位点/单体结合；这种结合与Mg++有协同作用。结果表明，PEP和Mg(2+)的生理浓度增加，而丙酮酸和Mg(2+)的浓度降低，二聚体、活性和脱磷Ei的含量减少。 在与Clore(美国国立卫生研究院)和Wang(奥马哈)实验室的合作下，继续通过核磁共振对临时技术秘书处各组分的溶液结构进行研究。通用的磷酸载体HPR与许多糖特异的IIA组分相互作用，解决了HPR与甘露醇IIA的细胞质结构域之间的复合体的结构(见参考文献2)。HPR上由螺旋1和2形成的凸面与由螺旋3、部分螺旋2和4以及β链2和3形成的IIA表面上的互补凹陷相互作用。大多数分子间接触是疏水的，在界面外围有少量静电相互作用。HPR和IIA的活性部位组氨酸非常接近，因此可以很容易地形成五配位的磷酸过渡态，而只需对骨架进行最小的扰动。与我们之前解决的两个HPR与伙伴蛋白(酶I和葡萄糖的IIA)的结构相比，尽管没有结构相似之处，但显示了共同的特征。因此，不同的底层结构元素可以形成形状和残基组成都相似的HPR结合表面。 解决了葡萄糖的IIA和葡萄糖的IICB的细胞质结构域之间的复合体的结构(见参考文献1)。界面是由IIA上的凹陷和IIB上的凹凸相互作用形成的，磷酰基给体和受体残基非常接近，埋在界面的中心。疏水的分子间相互作用由外围静电相互作用补充，这些相互作用涉及相互作用表面上正负电荷残基的交替分布。在不改变骨架构象的情况下，磷酰化过渡态很容易被调节，并且络合物的结构解释了IIA和IIB之间磷酰化转移的方向性。 葡萄糖IIA的N-末端结构域赋予蛋白质两面性，使其能够穿梭于细胞膜和细胞质之间。解决了与N-末端结构域相对应的合成肽的结构(见参考文献6)。在水中，结构是无序的，但在洗涤剂胶束中，肽的残基Phe3-Val10呈螺旋构象。在N-末端区域的四个赖氨酸中，只有两亲区的Lys5和Lys7与洗涤剂相互作用。显示了从洗涤剂到多肽的分子间NOs，支持N-末端结构域对膜的锚定功能。 葡萄糖的酶IIA在调节乳糖渗透酶中起直接作用；在半乳糖苷底物存在的情况下，去磷酸化的IIA直接与渗透酶结合。在与Kaback实验室(见参考文献5)的合作下，一个双Cys突变(Ile129Cys/Lys131Cys)被引入到渗透酶的螺旋IV中，位于细胞质IV/V中IIA结合部位附近以及在IV、V和VIII螺旋界面的底物结合部位附近。该突变体不再需要底物来与IIA结合；该突变体以高亲和力结合底物，但在跨细胞膜的所有易位模式中几乎完全缺陷。推测该双突变体为内向构象。 与Reddy实验室(见参考文献4)合作，从耻垢分枝杆菌中提纯了一种钙调蛋白样蛋白，并对其进行了部分测序；这些数据用于产生全长克隆，其DNA序列包含55个氨基酸的开放阅读框。在大肠杆菌中表达的耻垢分枝杆菌蛋白具有真核细胞钙调蛋白的特性：钙依赖刺激真核细胞的磷酸二酯酶，该酶可被钙调素拮抗剂三氟拉嗪抑制，并与抗牛脑钙调蛋白抗体反应。与蛋白质中存在的9个酸性氨基酸一致，与真核细胞钙调蛋白的4个结构域相比，存在一个假定的钙结合结构域，反映了该蛋白的较小尺寸(~6 kDa)。超速离心和质谱分析排除了钙促进纯化蛋白齐聚的可能性。

Structural and regulatory studies on protein components of the 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 in a Mg(2+)-requiring reaction to produce pyruvate. New studies, in collaboration with Ann Ginsburg (see Reference 3), utilized the inactive mutant of EI(H189A), in which alanine is substituted for the active site His189, where substrate binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP strongly promotes dimerization of EI(H189A), 5 mM pyruvate has the opposite effect. When the coupling between N- and C-terminal domain unfolding produced by PEP and Mg(2+)is inhibited by pyruvate, the dimerization constant for EI(H189A) decreases from >10 to the eighth power to <5x10 to the fifth power. PEP binds to one site/monomer of EI(H189A); this binding is synergistic with Mg++. The results of this study show that physiological concentrations of PEP and Mg(2+) increase, whereas pyruvate and Mg(2+) decrease the amount of dimeric, active, dephospho-EI. In collaboration with the laboratories of Clore (NIH) and Wang (Omaha), solution structures by NMR of components of the PTS have continued. The general phosphocarrier HPr interacts with numerous sugar-specific IIA components, The structure of the complex between HPr and the cytoplasmic domain of the IIA for mannitol was solved (see Reference 2). A convex surface on HPr, formed by helices 1 and 2, interacts with a complementary concave depression on the IIA surface formed by helix 3, portions of helices 2 and 4, and beta strands 2 and 3. The majority of intermolecular contacts are hydrophobic, with a small number of electrostatic interactions at the periphery of the interface. The active site histidines of HPr and IIA are in close proximity so that a pentacoordinate phosphoryl transition state can be readily formed with only minimal perturbation of the backbone. Comparison with our two previously solved structures of HPr with partner proteins (enzyme I and the IIA for glucose) shows common features despite absence of structural resemblances. Consequently, different underlying structural elements can form binding surfaces for HPr that are similar in both shape and residue composition. The structure of the complex between the IIA for glucose and the cytoplasmic domain of IICB for glucose was solved (see Reference 1). The interface is formed by interaction of a concave depression on IIA with a concave protrusion on IIB; the phosphoryl donor and acceptor residues are in close proximity and buried at the center of the interface. Hydrophobic intermolecular contacts are supplemented by peripheral electrostatic interactions involving an alternating distribution of positively and negatively charged residues on the interaction surfaces. A phosphoryl transition state is easily accommodated without any change in backbone conformation and the structure of the complex accounts for the directionality of phosphoryl transfer between IIA nd IIB. The N-terminal domain of glucose IIA confers amphitropism to the protein, allowing it to shuttle between the membrane and cytoplasm. The structure of a synthetic peptide corresponding to the N-terminal domain was solved (see Reference 6). In water, the structure is disordered, but in detergent micelles, residues Phe3-Val10 of the peptide adopt a helical conformation. Of the four lysines in the N-terminal domain, only Lys5 and Lys7 in the amphipathic region interact with detergent. Intermolecular NOEs from detergent to the peptide were shown, supporting an anchor function to the membrane for the N-terminal domain. The enzyme IIA for glucose plays a direct role in regulating lactose permease; dephosphorylated IIA binds directly to the permease in the presence of a galactoside substrate. In collaboration with the Kaback laboratory lab (see reference 5),a double Cys mutation (Ile129Cys/Lys131Cys) was introduced into helix IV of the permease near the IIA binding site in cytoplasmic loop IV/V and in the vicinity of the substrate binding site at the interfaces of helices IV, V and VIII. The mutant no longer requires substrate for IIA binding; this mutant binds substrate with high affinity, but is almost completely defective in all modes of translocation across the cytoplasmic membrane. It is suggested that the double mutant is locked in an inward-facing conformation. A calmodulin-like protein from Mycobacterium smegmatis was purified to homogeneity and partially sequenced, in collaboration with the Reddy lab (see Reference 4); these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino acid open reading frame. The M. smegmatis protein, expressed in E. coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin anatagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies. Consistent with the presence of nine acidic amino acids in the protein, there is one putative calcium-binding domain, compared to four such domains for eukaryotic calmodulin, reflecting the smaller size (~ 6 kDa) of this protein. Ultracentrifugal and mass spectral analysis excluded the possibility that calcium promotes oligomerization of the purified protein.