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首页> 外文期刊>Journal of Molecular Biology >Integrative modelling coupled with ion mobility mass spectrometry reveals structural features of the clamp loader in complex with single-stranded DNA binding protein
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Integrative modelling coupled with ion mobility mass spectrometry reveals structural features of the clamp loader in complex with single-stranded DNA binding protein

机译：集成建模与离子迁移质谱联用揭示了与单链DNA结合蛋白复合的钳式装载器的结构特征

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摘要

DNA polymerase III, a decameric 420-kDa assembly, simultaneously replicates both strands of the chromosome in Escherichia coli. A subassembly of this holoenzyme, the seven-subunit clamp loader complex, is responsible for loading the sliding clamp (β2) onto DNA. Here, we use structural information derived from ion mobility mass spectrometry (IM-MS) to build three-dimensional models of one form of the full clamp loader complex, γ3δδ′ψχ (254 kDa). By probing the interaction between the clamp loader and a single-stranded DNA (ssDNA) binding protein (SSB4) and by identifying two distinct conformational states, with and without ssDNA, we assemble models of ψχ-SSB4 (108 kDa) and the clamp loader-SSB4 (340 kDa) consistent with IM data. A significant increase in measured collision cross-section (~ 10%) of the clamp loader-SSB4 complex upon DNA binding suggests large conformational rearrangements. This DNA bound conformation represents the active state and, along with the presence of ψχ, stabilises the clamp loader-SSB 4 complex. Overall, this study of a large heteromeric complex analysed by IM-MS, coupled with integrative modelling, highlights the potential of such an approach to reveal structural features of previously unknown complexes of high biological importance.
机译：DNA聚合酶III是420 kDa的十聚体，可同时在大肠杆菌中复制两条染色体。该全酶的一个子组件，即七个亚基钳夹加载物复合体，负责将滑动钳（β2）加载到DNA上。在这里，我们使用从离子淌度质谱（IM-MS）得出的结构信息来构建全钳形装载机复合体γ3δδ'ψχ（254 kDa）一种形式的三维模型。通过探查夹具加载器与单链DNA（ssDNA）结合蛋白（SSB4）之间的相互作用，并通过识别带有和不带有ssDNA的两个不同的构象状态，我们构建了ψχ-SSB4（108 kDa）和夹具加载器的模型-SSB4（340 kDa）与IM数据一致。 DNA结合后，夹具装载器-SSB4复合物的测得碰撞截面的显着增加（〜10％），表明构象重排较大。该DNA结合的构象代表活性状态，并且与ψχ的存在一起稳定了钳夹加载物-SSB 4复合物。总体而言，这项对IM-MS分析的大型异聚体复合物的研究与综合建模相结合，凸显了这种方法揭示具有高度生物学重要性的先前未知复合物结构特征的潜力。

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