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首页> 外文期刊>Biochemistry >Analysis of the Structural and Functional Roles of Coupling Helices in the ATP-Binding Cassette Transporter MsbA through Enzyme Assays and Molecular Dynamics Simulations
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Analysis of the Structural and Functional Roles of Coupling Helices in the ATP-Binding Cassette Transporter MsbA through Enzyme Assays and Molecular Dynamics Simulations

机译：通过酶测定和分子动力学模拟分析ATP结合盒式磁带转运蛋白MSBA中偶联螺旋的结构和功能作用

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摘要

ATP-binding cassette (ABC) transporters are constructed from some common structural units: the highly conserved nucleotide-binding domains (NBDs), which work as a nucleotide-dependent engine for driving substrate transport, the diverse transmembrane domains (TMDs), which create the translocation pathway, and the coupling helices (CHs), which are located at the NBD?TMD interface. Although the CHs are believed to be essential for NBD?TMD communication, their roles remain unclear. In this study, we performed enzyme assays and molecular dynamics (MD) simulations of the ABC transporter MsbA and two MsbA mutants in which the amino acid residues of one of the CHs were mutated to alanines: (i) wild type (Wt), (ii) CH1 mutant (Mt1), and (iii) CH2 mutant (Mt2). The experiments show that the CH2 mutation decreases the ATPase activity (k_(cat)) compared with that of the Wt (a decrease of 32%), and a nearly equal degree of decrease in the ATP binding affinity (K_m) was observed for both Mt1 and Mt2. The MD simulations successfully accounted for several structural and dynamical origins for these experimental observations. In addition, on the basis of collective motion and morphing analyses, we propose that the reverserotational motions and noddinglike motions between the NBDs and TMDs are indispensable for the conformational transition between the inward- and outward-facing conformations. In particular, CH2 is significantly important for the occurrence of the noddinglike motion. These findings provide important insights into the structure?function relationship of ABC transporters.
机译：ATP结合盒（ABC）转运蛋白由一些常见的结构单元构成：高度保守的核苷酸结合结构域（NBD），其作为用于驱动基材传输的核苷酸依赖的发动机，这些跨膜结构域（TMDS）是创造的易位途径和耦合螺旋（CHS）位于NBDαTMD接口。虽然CHS被认为是NBD的必要条件，但他们的角色仍然不清楚。在该研究中，我们进行了ABC转运蛋白MSBA的酶测定和分子动力学（MD）模拟，其中将其中一个CH的氨基酸残基突变为丙氨酸：（i）野生型（WT），（ II）CH1突变体（MT1）和（III）CH2突变体（MT2）。实验表明，与WT（32％的降低）相比，CH2突变降低了ATP酶活性（K_（猫）），并且对于两者都观察到接近ATP结合亲和力（K_M）的几乎相同程度的降低MT1和MT2。 MD模拟成功地占这些实验观察的几种结构和动态起源。此外，在集体运动和变形分析的基础上，我们提出了Nbds和Tmds之间的反向主动动动动动力和点状运动对于内侧和向外构象之间的构象过渡是必不可少的。特别是，CH2对于出现点状运动的发生显着重要。这些发现提供了对结构的重要见解？ABC运输车的功能关系。

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  • 来源
    《Biochemistry》 |2014年第26期|共12页
  • 作者

    Tadaomi Furuta; Tomohiro Yamaguchi; Hiroaki Kato; Minoru Sakurai;

  • 作者单位

    Center for Biological Resources and Informatics Tokyo Institute of Technology B-62 4259 Nagatsuta-cho Midori-ku Yokohama 226-8501 Japan;

    Graduate School of Pharmaceutical Sciences Kyoto University 46-29 Yoshida-Shimo-Adachi-cho Sakyo-ku Kyoto 606-8501 Japan;

    Graduate School of Pharmaceutical Sciences Kyoto University 46-29 Yoshida-Shimo-Adachi-cho Sakyo-ku Kyoto 606-8501 Japan;

    Center for Biological Resources and Informatics Tokyo Institute of Technology B-62 4259 Nagatsuta-cho Midori-ku Yokohama 226-8501 Japan;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

    Analysis; Coupling; Simulations;

    机译：分析;耦合;模拟;

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