Effects of targeting signal mutations in a mitochondrial presequence on the spatial distribution of the conformational ensemble in the binding site of Tom20
Effects of targeting signal mutations in a mitochondrial presequence on the spatial distribution of the conformational ensemble in the binding site of Tom20
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DOI：
10.1002/pro.4433
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发表时间：
2022-09
期刊：
影响因子：
8
通讯作者：
Xiling Han;N. Maita;A. Shimada;D. Kohda
中科院分区：
文献类型：
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作者：
Xiling Han;N. Maita;A. Shimada;D. Kohda
20‐kDa的TOM（线粒体外膜转位酶）亚基Tom20是线粒体蛋白质输入途径的第一个受体。Tom20识别嵌入在成熟线粒体蛋白上的线粒体靶向信号，作为N端延伸。因此，约1000种不同的线粒体蛋白被分类到线粒体基质中，并与非线粒体蛋白区分开来。我们之前报道了MPRIDE （multiple partial recognition in dynamic equilibrium）机制来解释Tom20混杂识别序列的结构基础。在绑定状态下的序列的每个位姿中识别目标信号特征的子集，并在位姿之间的动态平衡中对所有特征进行集体识别。在这里，我们改变了靶向信号中疏水侧链的体积，同时保持了结合亲和力。我们将突变序列连接到Tom20的结合位点，并将它们放置在晶格中创建的晶体无接触空间（CCFS）中。突变序列的空间分布在X射线晶体学获得的低通滤波差分图中以模糊的电子密度表现出来。突变的序列在结合状态下改变了它们的位置，以适应更大的侧链，从而为MPRIDE机制在Tom20的混杂识别中使用提供了积极的证据。
The 20‐kDa TOM (translocase of outer mitochondrial membrane) subunit, Tom20, is the first receptor of the protein import pathway into mitochondria. Tom20 recognizes the mitochondrial targeting signal embedded in the presequences attached to mature mitochondrial proteins, as an N‐terminal extension. Consequently, ~1,000 different mitochondrial proteins are sorted into the mitochondrial matrix, and distinguished from non‐mitochondrial proteins. We previously reported the MPRIDE (multiple partial recognitions in dynamic equilibrium) mechanism to explain the structural basis of the promiscuous recognition of presequences by Tom20. A subset of the targeting signal features is recognized in each pose of the presequence in the binding state, and all of the features are collectively recognized in the dynamic equilibrium between the poses. Here, we changed the volumes of the hydrophobic side chains in the targeting signal, while maintaining the binding affinity. We tethered the mutated presequences to the binding site of Tom20 and placed them in the crystal contact‐free space (CCFS) created in the crystal lattice. The spatial distributions of the mutated presequences were visualized as smeared electron densities in the low‐pass filtered difference maps obtained by X‐ray crystallography. The mutated presequence ensembles shifted their positions in the binding state to accommodate the larger side chains, thus providing positive evidence supporting the use of the MPRIDE mechanism in the promiscuous recognition by Tom20.