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首页> 外文期刊>Biochemistry >Crystallographic Snapshots of Tom20-Mitochondrial Presequence Interactions with Disulfide-Stabilized Peptides
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Crystallographic Snapshots of Tom20-Mitochondrial Presequence Interactions with Disulfide-Stabilized Peptides

机译：Tom20线粒体与二硫键稳定的肽相互作用的晶体快照。

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Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The Tom20 protein, residing on the mitochondrial surface, recognizes the N-terminal presequences of precursor proteins. We previously determined the crystal structures of the Tom20 presequence complex. The successful crystallization involved tethering the presequence to Tom20 through an intermolecular disulfide bond with an optimized linker. In this work, we assessed the tethering method. The intermolecular disulfide bond was cleaved in crystal with a reducing agent. The pose (i.e., conformation and position) of the presequence was identical to the previously determined pose. In another experiment, a longer linker than the optimized length was used for the tethering. The perturbation of the tether changed the pose slightly, but the interaction mode was preserved. These results argue against the forced interaction of the presequence by its covalent attachment to Tom20. Second, as an alternative method referred to as "molecular stiffening", we introduced a disulfide bond within the presequence peptide to restrict the freedom of the peptide in the unbound states. One presequence analogue exhibited over 100-fold higher affinity than its linear counterpart and generated cocrystals with Tom20. One of the two crystallographic snapshots revealed a known pose previously determined by the tethering method, and the other snapshot depicted a new pose. These results confirmed and extended the dynamic, multiple bound state model of the Tom20-presequence interactions and also demonstrated the validity of the molecular tethering and stiffening techniques in studies of transient protein-peptide interactions.
机译：大多数线粒体蛋白在细胞质中合成并导入线粒体。驻留在线粒体表面上的Tom20蛋白可以识别前体蛋白的N端序列。我们先前确定了Tom20序列前复合物的晶体结构。成功的结晶包括通过分子间二硫键和优化的连接子将序列连接到Tom20。在这项工作中，我们评估了网络共享方法。用还原剂将分子间二硫键在晶体中裂解。序列的姿势（即构象和位置）与先前确定的姿势相同。在另一个实验中，将比最佳长度更长的接头用于系链。系绳的摄动稍微改变了姿势，但保留了交互方式。这些结果与共价附着于Tom20的先决序列的强制相互作用相反。其次，作为一种称为“分子硬化”的替代方法，我们在预序列肽中引入了二硫键以限制未结合状态下的肽自由度。一种预序列类似物显示出比其线性对应物高100倍以上的亲和力，并与Tom20产生共晶体。两个晶体快照中的一个快照显示了以前通过系留方法确定的已知姿势，另一个快照描述了一个新的姿势。这些结果证实并扩展了Tom20-presequence相互作用的动态，多重结合状态模型，并且还证明了分子束缚和强化技术在瞬时蛋白-肽相互作用研究中的有效性。

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