Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, United States of America.
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, United States of America.
PLoS One. 2019 May 2;14(5):e0213423. doi: 10.1371/journal.pone.0213423. eCollection 2019.
Pore-forming proteins (PFPs) represent a functionally important protein family, that are found in organisms from viruses to humans. As a major branch of PFPs, bacteria pore-forming toxins (PFTs) permeabilize membranes and usually cause the death of target cells. E. coli hemolysin ClyA is the first member with the pore complex structure solved among α-PFTs, employing α-helices as transmembrane elements. ClyA is proposed to form pores composed of various numbers of protomers. With high-resolution cryo-EM structures, we observe that ClyA pore complexes can exist as newly confirmed oligomers of a tridecamer and a tetradecamer, at estimated resolutions of 3.2 Å and 4.3 Å, respectively. The 2.8 Å cryo-EM structure of a dodecamer dramatically improves the existing structural model. Structural analysis indicates that protomers from distinct oligomers resemble each other and neighboring protomers adopt a conserved interaction mode. We also show a stabilized intermediate state of ClyA during the transition process from soluble monomers to pore complexes. Unexpectedly, even without the formation of mature pore complexes, ClyA can permeabilize membranes and allow leakage of particles less than ~400 Daltons. In addition, we are the first to show that ClyA forms pore complexes in the presence of cholesterol within artificial liposomes. These findings provide new mechanistic insights into the dynamic process of pore assembly for the prototypical α-PFT ClyA.
孔形成蛋白（PFPs）是一个功能重要的蛋白质家族，在从病毒到人类的生物体中都有发现。作为 PFPs 的一个主要分支，细菌孔形成毒素（PFTs）可使细胞膜穿孔，通常导致靶细胞死亡。大肠杆菌溶血素 ClyA 是 α-PFTs 中第一个解决了孔复合物结构的成员，其使用α-螺旋作为跨膜元件。ClyA 被认为可以形成由各种数量的原体组成的孔。通过高分辨率的冷冻电镜结构，我们观察到 ClyA 孔复合物可以以新确认的十三聚体和十四聚体的寡聚体形式存在，分别估计分辨率为 3.2Å 和 4.3Å。2.8Å 的十二聚体冷冻电镜结构极大地改进了现有的结构模型。结构分析表明，来自不同寡聚体的原体彼此相似，相邻的原体采用保守的相互作用模式。我们还展示了 ClyA 在从可溶性单体向孔复合物转变过程中的稳定中间状态。出乎意料的是，即使没有成熟孔复合物的形成，ClyA 也可以使膜穿孔，并允许小于~400 道尔顿的颗粒泄漏。此外，我们是第一个表明 ClyA 在人工脂质体中存在胆固醇的情况下形成孔复合物的人。这些发现为原型α-PFT ClyA 的孔组装动态过程提供了新的机制见解。
