Nanomechanical and Thermophoretic Analyses of the Nucleotide-Dependent Interactions between the AAA(+) Subunits of Magnesium Chelatase.
Nanomechanical and Thermophoretic Analyses of the Nucleotide-Dependent Interactions between the AAA(+) Subunits of Magnesium Chelatase.
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DOI：
10.1021/jacs.6b02827
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发表时间：
2016-05-25
影响因子：
15
通讯作者：
Hunter CN
中科院分区：
文献类型：
--
作者：
Adams NB;Vasilev C;Brindley AA;Hunter CN
在叶绿素生物合成中，镁螯合酶复合物催化Mg 2+离子插入原卟啉IX。在此之前，三个亚基中的两个，AAA+蛋白ChlI和ChlD，形成ChlID-MgATP复合物。我们使用微尺度热泳直接测定解离常数的I-D亚基从集胞藻，并表明，形成的ChlID-MgADP复合物，介导的精氨酸指和传感器II结构域ChlD，是必要的组装的催化活性ChlHID-MgATP复合物。ChlD的N-末端AAA+结构域对于复合物的形成是必需的，但是在ChlD的C-末端整联蛋白结构域不存在的情况下保留了一些稳定性，特别是如果保留了插入的聚脯氨酸接头区域。单分子力谱（SMFS）用于确定在单分子水平上稳定ChlID-MgADP复合物形成的因素：ChlD以两种不同的方向附着在原子力显微镜（AFM）探针上，ChlI亚基被束缚在二氧化硅表面;亚基相互作用的概率在MgADP存在下增加了一倍以上，并且我们表明ChlD的N-末端AAA+结构域介导了这一过程，与微尺度热泳数据一致。未结合数据的分析揭示了形成单个ChlID-MgADP复合物的最可能的相互作用力为约109 pN。这些实验为理解镁螯合酶复合物的组装和功能提供了定量基础。
In chlorophyll biosynthesis, the magnesium chelatase enzyme complex catalyzes the insertion of a Mg2+ ion into protoporphyrin IX. Prior to this event, two of the three subunits, the AAA+ proteins ChlI and ChlD, form a ChlID–MgATP complex. We used microscale thermophoresis to directly determine dissociation constants for the I-D subunits from Synechocystis, and to show that the formation of a ChlID–MgADP complex, mediated by the arginine finger and the sensor II domain on ChlD, is necessary for the assembly of the catalytically active ChlHID–MgATP complex. The N-terminal AAA+ domain of ChlD is essential for complex formation, but some stability is preserved in the absence of the C-terminal integrin domain of ChlD, particularly if the intervening polyproline linker region is retained. Single molecule force spectroscopy (SMFS) was used to determine the factors that stabilize formation of the ChlID–MgADP complex at the single molecule level; ChlD was attached to an atomic force microscope (AFM) probe in two different orientations, and the ChlI subunits were tethered to a silica surface; the probability of subunits interacting more than doubled in the presence of MgADP, and we show that the N-terminal AAA+ domain of ChlD mediates this process, in agreement with the microscale thermophoresis data. Analysis of the unbinding data revealed a most probable interaction force of around 109 pN for formation of single ChlID–MgADP complexes. These experiments provide a quantitative basis for understanding the assembly and function of the Mg chelatase complex.