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首页> 外文期刊>Biochemistry >Mutations That Probe the Cooperative Assembly of O6-Alkylguanine-DNA Alkyltransferase Complexes
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Mutations That Probe the Cooperative Assembly of O6-Alkylguanine-DNA Alkyltransferase Complexes

机译：探测O6-烷基鸟嘌呤-DNA烷基转移酶复合物协同装配的突变

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O6n-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6n-alkylguanine and O4n-nalkylthymine adducts present in DNA that has been exposed to alkylating agents. AGT binds DNAncooperatively, and models of cooperative complexes predict that residues 1-7 of one protein molecule andnresidues 163-169 of a neighboring protein are closely juxtaposed. To test these models, we used directednmutagenesis to substitute triplets of alanine for triplets of native residues across these two sequences. Six ofneight designedmutants expressed AGT at detectable levels. Allmutant AGTs that were expressed were foldedncompactly, bound DNA with stoichiometries equivalent to that of the wild-type protein, and were able tonprotect Escherichia coli to varying degrees from the potent alkylating agent N-methyl-N -nitro-N-nitroso-nguanidine (MNNG). All mutations attenuated DNA binding cooperativity, but unexpectedly, they alsonreduced the affinity ofAGT forDNA. This suggests that the protein-protein and protein-DNAinteractionsnof AGT are strongly coupled.When normalized for differences in AGT expression, cells expressing mutantsnKDC(3-5)-AAA, DCE(4-6)-AAA, and KEW(165-167)-AAA were significantly more susceptible tonMNNG than wild-type cells. This is the first evidence, to the best of our knowledge, of a role for residuesnat the protein-protein interface and, by implication, cooperative protein-protein interactions in the cell-nprotective mechanisms of AGT.
机译：O6n-烷基鸟嘌呤-DNA烷基转移酶（AGT）修复暴露于烷基化剂的DNA中存在的诱变O6n-烷基鸟嘌呤和O4n-n烷基胸腺嘧啶加合物。 AGT可合作地结合DNA，合作复合物模型预测一个蛋白质分子的残基1-7和邻近蛋白质的残基163-169紧密并列。为了测试这些模型，我们使用定向诱变法将丙氨酸的三联体替换为这两个序列中天然残基的三联体。 8个设计突变体中有6个以可检测的水平表达了AGT。将表达的所有突变AGT紧密折叠，以与野生型蛋白相同的化学计量比结合DNA，并且能够从有效的烷基化剂N-甲基-N-硝基-N-亚硝基-胍（不同程度地）保护大肠杆菌。 MNNG）。所有突变均减弱了DNA结合的协同作用，但出乎意料的是，它们也降低了AGT对DNA的亲和力。这表明AGT的蛋白质-蛋白质和蛋白质-DNA相互作用紧密耦合。当针对AGT表达差异进行标准化时，表达突变体的细胞nKDC（3-5）-AAA，DCE（4-6）-AAA和KEW（165-167） ）-AAA对tonMNNG的敏感性明显高于野生型细胞。据我们所知，这是残留蛋白在蛋白质-蛋白质界面中的作用的首次证据，并暗示了蛋白质-蛋白质相互作用在AGT的细胞保护机制中的作用。

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    《Biochemistry》 |2011年第10期|p.1590-1598|共9页
  • 作者

    Claire A. Adams and Michael G. Fried;

  • 作者单位

    ‡Center for Structural Biology, Department of Molecular and Cellular Biochemistry and §Department of Microbiology,Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky 40536, United States;

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