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首页> 外文期刊>Journal of Molecular Biology >NMR structure of cysteinyl-phosphorylated enzyme IIB of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.
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NMR structure of cysteinyl-phosphorylated enzyme IIB of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

机译：大肠杆菌的N，N'-二乙酰基壳二糖特异性磷酸烯醇丙酮酸依赖性磷酸转移酶系统的半胱氨酰磷酸化酶IIB的NMR结构。

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摘要

The determination by NMR of the solution structure of the phosphorylated enzyme IIB (P-IIB(Chb)) of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli is presented. Most of the backbone and side-chain resonances were assigned using a variety of mostly heteronuclear NMR experiments. The remaining resonances were assigned with the help of the structure calculations.NOE-derived distance restraints were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In addition, combinations of ambiguous restraints were used to resolve ambiguities in the NOE assignments. By combining sets of ambiguous and unambiguous restraints into new ambiguous restraints, an error function was constructed that was less sensitive to information loss caused by assignment uncertainties. The final set of structures had a pairwise rmsd of 0.59 A and 1.16 A for the heavy atoms of the backbone and side-chains, respectively.Comparing the P-IIB(Chb) solution structure with the previously determined NMR and X-ray structures of the wild-type and the Cys10Ser mutant shows that significant differences between the structures are limited to the active-site region. The phosphoryl group at the active-site cysteine residue is surrounded by a loop formed by residues 10 through 16. NOE and chemical shift data suggest that the phosphoryl group makes hydrogen bonds with the backbone amide protons of residues 12 and 15. The binding mode of the phosphoryl group is very similar to that of the protein tyrosine phosphatases. The differences observed are in accordance with the presumption that IIB(Chb) has to be more resistant to hydrolysis than the protein tyrosine phosphatases. We propose a proton relay network by which a transfer occurs between the cysteine SH proton and the solvent via the hydroxyl group of Thr16. Copyright 2001 Academic Press.
机译：通过NMR确定了N，N′-二乙酰基壳二糖特异性磷酸烯醇丙酮酸依赖性磷酸转移酶系统的磷酸化酶IIB（P-IIB（Chb））的溶液结构。大多数主链和侧链共振是通过各种主要是异核NMR实验指定的。其余的共振在结构计算的帮助下进行分配。NOE衍生的距离约束用于距离几何计算，然后进行分子动力学和模拟退火方案。此外，模糊约束的组合被用来解决NOE任务中的歧义。通过将模糊的和明确的约束集组合为新的模糊的约束，构建了一个误差函数，该误差函数对分配不确定性导致的信息丢失不那么敏感。最后一组结构的主链和侧链重原子的成对均方根均方根（rmsd）分别为0.59 A和1.16A。将P-IIB（Chb）溶液结构与先前确定的NMR和X射线结构进行比较野生型和Cys10Ser突变体表明，结构之间的显着差异仅限于活性位点区域。活性位点半胱氨酸残基上的磷酰基被残基10至16形成的环包围。NOE和化学位移数据表明，磷酰基与残基12和15的骨架酰胺质子形成氢键。磷酸基团与蛋白质酪氨酸磷酸酶非常相似。观察到的差异是根据这样的假设，即IIB（Chb）必须比蛋白酪氨酸磷酸酶更耐水解。我们提出了一种质子中继网络，通过该网络，半胱氨酸SH质子和溶剂之间通过Thr16的羟基发生转移。版权所有2001学术出版社。

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