The Biology Of Sugar Transport in E Coli

大肠杆菌中糖运输的生物学

• 批准号: 6815637
• 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
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6815637
• 关键词: Escherichia coli; Mycoplasma; bacterial genetics; bacterial proteins; calmodulin; carbohydrate transport; enzyme complex; enzyme mechanism; enzyme structure; gene expression; lactose; microorganism metabolism; nuclear magnetic resonance spectroscopy; permease; phosphodiesterases; phosphorylation; phosphotransferases; protein protein interaction; protein structure function

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.

对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））的蛋白质成分的结构和调控研究仍在继续。 PTS 的第一个组分（酶 I，EI）在需要 Mg(2+) 的反应中被活性位点组氨酸上的磷酸烯醇丙酮酸 (PEP) 磷酸化，产生丙酮酸。与 Ann Ginsburg 合作的新研究（参见参考文献 3）利用了 EI(H189A) 的无活性突变体，其中丙氨酸取代了活性位点 His189，其中底物结合效应可以与磷酸化效应分开。 1 mM PEP 强烈促进 EI(H189A) 二聚化，而 5 mM 丙酮酸则具有相反的作用。当 PEP 和 Mg(2+) 产生的 N 端和 C 端结构域解折叠之间的偶联被丙酮酸抑制时，EI(H189A) 的二聚常数从 >10 的八次方降低到 <5x10 的五次方。 PEP 与 EI(H189A) 的一个位点/单体结合；该结合与 Mg++ 具有协同作用。本研究的结果表明，PEP 和 Mg(2+) 的生理浓度增加，而丙酮酸和 Mg(2+) 减少二聚体、活性去磷酸-EI 的量。 与 Clore (NIH) 和 Wang (Omaha) 实验室合作，继续通过 NMR 分析 PTS 组分的溶液结构。一般磷酸载体HPr与众多糖特异性IIA组分相互作用，解决了HPr与甘露醇IIA胞质结构域之间的复合物结构（参见参考文献2）。 HPr 上由螺旋 1 和 2 形成的凸面与 IIA 表面上由螺旋 3、螺旋 2 和 4 的部分以及 β 链 2 和 3 形成的互补凹面相互作用。大多数分子间接触是疏水性的，在界面外围有少量静电相互作用。 HPr 和 IIA 的活性位点组氨酸非常接近，因此只需对主链进行最小的扰动即可轻松形成五配位磷酰基过渡态。与我们之前解决的两个 HPr 结构和伴侣蛋白（酶 I 和葡萄糖 IIA）的比较显示出共同的特征，尽管没有结构相似性。因此，不同的底层结构元件可以形成形状和残基组成相似的 HPr 结合表面。 解决了葡萄糖的 IIA 和葡萄糖的 IICB 的胞质结构域之间的复合物结构（参见参考文献 1）。该界面是由IIA上的凹入凹陷与IIB上的凹入凸起相互作用形成的；磷酰基供体和受体残基非常接​​近并埋在界面的中心。疏水性分子间接触由外围静电相互作用补充，涉及相互作用表面上带正电和带负电的残基的交替分布。磷酰基过渡态很容易适应，主链构象不会发生任何变化，并且复合物的结构解释了 IIA 和 IIB 之间磷酰基转移的方向性。 葡萄糖 IIA 的 N 末端结构域赋予蛋白质两向性，使其能够在膜和细胞质之间穿梭。解析了对应于 N 末端结构域的合成肽的结构（参见参考文献 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 端结构域的四个赖氨酸中，只有两亲性区域的 Lys5 和 Lys7 与去污剂相互作用。显示了从去垢剂到肽的分子间 NOE，支持 N 端结构域对膜的锚定功能。 葡萄糖酶 IIA 在调节乳糖通透酶中起着直接作用；在半乳糖苷底物存在的情况下，去磷酸化的 IIA 直接与通透酶结合。与 Kaback 实验室合作（参见参考文献 5），将双 Cys 突变 (Ile129Cys/Lys131Cys) 引入通透酶的螺旋 IV 中，靠近细胞质环 IV/V 中的 IIA 结合位点以及螺旋 IV、V 和 VIII 界面处的底物结合位点附近。突变体不再需要 IIA 结合底物；该突变体以高亲和力结合底物，但在跨细胞质膜的所有易位模式中几乎完全有缺陷。这表明双突变体被锁定在向内的构象中。 与 Reddy 实验室合作，将耻垢分枝杆菌中的类钙调蛋白纯化至均质并进行部分测序（参见参考文献 4）；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表现出真核钙调蛋白的特性：钙依赖性刺激真核磷酸二酯酶，该酶被钙调蛋白拮抗剂三氟拉嗪抑制，并与抗牛脑钙调蛋白抗体发生反应。与该蛋白质中存在 9 个酸性氨基酸一致，存在一个推定的钙结合结构域，而真核钙调蛋白有 4 个这样的结构域，这反映出该蛋白质的尺寸较小（约 6 kDa）。超速离心和质谱分析排除了钙促进纯化蛋白质寡聚化的可能性。