A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex
A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex
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DOI：
10.1371/journal.pbio.3000475
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发表时间：
2019-10-01
期刊：
影响因子：
9.8
通讯作者：
Bradley, Peter John
中科院分区：
文献类型：
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作者：
Choi, Charles Paul;Moon, Andy Seong;Bradley, Peter John
弓形虫内膜复合体（IMC）是弓形虫体内重要的细胞器，参与寄生虫的运动和复制。IMC位于寄生虫的质膜之下，由膜和细胞骨架组成。虽然IMC的蛋白质组成正在得到更好的理解，使细胞器的正常功能的蛋白质-蛋白质协会仍然在很大程度上未知。确定IMC细胞骨架网络中的蛋白质相互作用特别具有挑战性，因为破坏细胞骨架需要破坏蛋白质复合物的条件。为了解决这个问题，我们展示了应用光反应性非天然氨基酸（UAA）交联系统来捕获天然细胞内环境中的蛋白质相互作用。除了识别结合伴侣，UAA方法还绘制了用于交联的诱饵蛋白的结合界面，提供了相互作用蛋白的结构信息。我们将这种技术应用于必需的IMC蛋白ILP 1，并证明其C-末端卷曲螺旋结构域的不同区域与肺泡蛋白IMC 3和IMC 6以及IMC 27交联。我们还表明，IMC 3的C-末端结构域和IMC 6的N-末端结构域是必要的结合ILP 1，进一步映射ILP 1和细胞骨架之间的相互作用。总之，这项研究开发了一种新的方法来研究弓形虫蛋白质-蛋白质相互作用，并提供了第一个深入了解顶复体IMC的细胞骨架网络的架构。
The Toxoplasma gondii inner membrane complex (IMC) is an important organelle involved in parasite motility and replication. The IMC resides beneath the parasite's plasma membrane and is composed of both membrane and cytoskeletal components. Although the protein composition of the IMC is becoming better understood, the protein-protein associations that enable proper functioning of the organelle remain largely unknown. Determining protein interactions in the IMC cytoskeletal network is particularly challenging, as disrupting the cytoskeleton requires conditions that disrupt protein complexes. To circumvent this problem, we demonstrate the application of a photoreactive unnatural amino acid (UAA) crosslinking system to capture protein interactions in the native intracellular environment. In addition to identifying binding partners, the UAA approach maps the binding interface of the bait protein used for crosslinking, providing structural information of the interacting proteins. We apply this technology to the essential IMC protein ILP1 and demonstrate that distinct regions of its C-terminal coiled-coil domain crosslink to the alveolins IMC3 and IMC6, as well as IMC27. We also show that the IMC3 C-terminal domain and the IMC6 N-terminal domain are necessary for binding to ILP1, further mapping interactions between ILP1 and the cytoskeleton. Together, this study develops a new approach to study protein-protein interactions in Toxoplasma and provides the first insight into the architecture of the cytoskeletal network of the apicomplexan IMC.