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首页> 外文期刊>Journal of Molecular Biology >Mutational Analysis of the Uracil DNA Glycosylase Inhibitor Protein and Its Interaction with Escherichia coli Uracil DNA Glycosylase.
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Mutational Analysis of the Uracil DNA Glycosylase Inhibitor Protein and Its Interaction with Escherichia coli Uracil DNA Glycosylase.

机译：尿嘧啶DNA糖基化酶抑制剂蛋白的突变分析及其与大肠杆菌尿嘧啶DNA糖基化酶的相互作用。

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摘要

Uracil DNA glycosylase inhibitor (Ugi), a protein of 9.4kDa consists of a five-stranded antiparallel beta sheet flanked on either side by single alpha helices, forms an exclusive complex with uracil DNA glycosylases (UDGs) that is stable in 8M urea. We report on the mutational analysis of various structural elements in Ugi, two of which (hydrophobic pocket and the beta1 edge) establish key interactions with Escherichia coli UDG. The point mutations in helix alpha1 (amino acid residues 3-14) do not affect the stability of the UDG-Ugi complexes in urea. And, while the complex of the DeltaN13 mutant with UDG is stable in only approximately 4M urea, its overall structure and thermostability are maintained. The identity of P37, stacked between P26 and W68, was not important for the maintenance of the hydrophobic pocket or for the stability of the complex. However, the M24K mutation at the rim of the hydrophobic pocket lowered the stability of the complex in 6M urea. On the other hand, non-conservative mutations E49G, D61G (cancels the only ionic interaction with UDG) and N76K, in three of the loops connecting the beta strands, conferred no such phenotype. The L23R and S21P mutations (beta1 edge) at the UDG-Ugi interface, and the N35D mutation far from the interface resulted in poor stability of the complex. However, the stability of the complexes was restored in the L23A, S21T and N35A mutations. These analyses and the studies on the exchange of Ugi mutants in preformed complexes with the substrate or the native Ugi have provided insights into the two-step mechanism of UDG-Ugi complex formation. Finally, we discuss the application of the Ugi isolates in overproduction of UDG mutants, toxic to cells.
机译：尿嘧啶DNA糖基化酶抑制剂（Ugi）是一种9.4kDa的蛋白质，由一个五链的反平行β片层组成，两侧均带有单个alpha螺旋，与尿嘧啶DNA糖基化酶（UDG）形成独家复合物，该尿苷在8M尿素中稳定。我们报告了在Ugi中的各种结构元素的突变分析，其中两个（疏水口袋和beta1边缘）建立了与大肠杆菌UDG的关键相互作用。螺旋α1（氨基酸残基3-14）中的点突变不影响UDG-Ugi复合物在尿素中的稳定性。并且，虽然DeltaN13突变体与UDG的复合物仅在约4M尿素中稳定，但其总体结构和热稳定性得以保持。堆叠在P26和W68之间的P37的身份对于维持疏水袋或配合物的稳定性并不重要。但是，疏水口袋边缘的M24K突变降低了复合物在6M尿素中的稳定性。另一方面，在连接β链的三个环中，非保守突变E49G，D61G（取消了与UDG的唯一离子相互作用）和N76K没有赋予这种表型。 UDG-Ugi界面处的L23R和S21P突变（beta1边缘）以及远离该界面的N35D突变导致复合物的稳定性较差。但是，复合物的稳定性在L23A，S21T和N35A突变中得以恢复。这些分析和对预先形成的复合物中的Ugi突变体与底物或天然Ugi交换的研究提供了对UDG-Ugi复合物形成两步机制的见解。最后，我们讨论了Ugi分离株在对细胞有毒的UDG突变体的过量生产中的应用。

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