Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia.
J Biol Chem. 2011 Jan 28;286(4):2946-55. doi: 10.1074/jbc.M110.169417. Epub 2010 Oct 2.
Perforin (PFN) is a pore-forming protein produced by cytotoxic lymphocytes that aids in the clearance of tumor or virus-infected cells by a mechanism that involves the formation of transmembrane pores. The properties of PFN pores and the mechanism of their assembly remain unclear. Here, we studied pore characteristics by functional and structural methods to show that perforin forms pores more heterogeneous than anticipated. Planar lipid bilayer experiments indicate that perforin pores exhibit a broad range of conductances, from 0.15 to 21 nanosiemens. In comparison with large pores that possessed low noise and remained stably open, small pores exhibited high noise and were very unstable. Furthermore, the opening step and the pore size were dependent on the lipid composition of the membrane. The heterogeneity in pore sizes was confirmed with cryo-electron microscopy and showed a range of sizes matching that observed in the conductance measurements. Furthermore, two different membrane-bound PFN conformations were observed, interpreted as pre-pore and pore states of the protein. The results collectively indicate that PFN forms heterogeneous pores through a multistep mechanism and provide a new paradigm for understanding the range of different effects of PFN and related membrane attack complex/perforin domain proteins observed in vivo and in vitro.
穿孔素（PFN）是一种由细胞毒性淋巴细胞产生的形成孔道的蛋白，有助于通过形成跨膜孔道的机制清除肿瘤或病毒感染的细胞。PFN 孔道的特性及其组装的机制仍不清楚。在这里，我们通过功能和结构方法研究了孔的特性，结果表明穿孔素形成的孔比预期的更不均匀。平面脂质双层实验表明，穿孔素孔具有从 0.15 到 21 纳西门子的广泛传导率。与具有低噪声且保持稳定开放的大孔相比，小孔具有高噪声且非常不稳定。此外，开口步骤和孔径取决于膜的脂质组成。用冷冻电子显微镜证实了孔径的不均匀性，并显示出与电导测量中观察到的大小范围相匹配的一系列大小。此外，还观察到了两种不同的膜结合 PFN 构象，解释为蛋白的预孔和孔状态。这些结果共同表明，PFN 通过多步机制形成不均匀的孔道，并为理解体内和体外观察到的 PFN 和相关膜攻击复合物/穿孔素结构域蛋白的不同作用范围提供了新的范例。
