Crystallographic snapshots of Tom20-mitochondrial presequence interactions with disulfide-stabilized peptides
Crystallographic snapshots of Tom20-mitochondrial presequence interactions with disulfide-stabilized peptides
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Tom20 线粒体前序列与二硫键稳定肽相互作用的晶体快照
DOI：
10.1021/bi200470x
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发表时间：
2011
期刊：
影响因子：
2.9
通讯作者：
Kohda D.
中科院分区：
文献类型：
--
作者：
Saitoh T;Igura M;Miyazaki Y;Ose T;Maita N;Kohda D.
大多数线粒体蛋白质在细胞质中合成并输入线粒体。Tom 20蛋白位于线粒体表面，识别前体蛋白的N-末端前序列。我们以前确定的晶体结构的Tom 20前序复合体。成功的结晶涉及通过分子间二硫键与优化的接头将前序列与Tom 20连接。在这项工作中，我们评估了拴系方法。用还原剂使晶体中的分子间二硫键断裂。姿势（即，构象和位置）与先前确定的姿态相同。在另一个实验中，使用比优化长度更长的接头用于系链。系绳的扰动略微改变了姿态，但保留了相互作用模式。这些结果反对其共价连接到Tom 20的前序列的强制相互作用。其次，作为一种被称为“分子硬化”的替代方法，我们在前序列肽中引入了二硫键以限制肽在未结合状态下的自由。一种前序列类似物表现出比其线性对应物高100倍以上的亲和力，并与Tom 20产生共晶。两个晶体学快照中的一个揭示了先前通过系链方法确定的已知姿态，另一个快照描绘了新姿态。这些结果证实并扩展了Tom 20-前序列相互作用的动态多束缚态模型，也证明了分子束缚和硬化技术在瞬时蛋白质-肽相互作用研究中的有效性。
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 cleavedin crystalwith 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.