State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Molecules. 2021 Aug 27;26(17):5194. doi: 10.3390/molecules26175194.
Necroptosis is a type of programmed cell death executed through the plasma membrane disruption by mixed lineage kinase domain-like protein (MLKL). Previous studies have revealed that an N-terminal four-helix bundle domain (NBD) of MLKL is the executioner domain for the membrane permeabilization, which is auto-inhibited by the first brace helix (H6). After necroptosis initiation, this inhibitory brace helix detaches and the NBD can integrate into the membrane, and hence leads to necroptotic cell death. However, how the NBD is released and induces membrane rupture is poorly understood. Here, we reconstituted MLKL into membrane mimetic bicelles and observed the structure disruption and membrane release of the first brace helix that is regulated by negatively charged phospholipids in a dose-dependent manner. Using molecular dynamics simulation we found that the brace region in an isolated, auto-inhibited MLKL becomes intrinsically disordered in solution after 7 ns dynamic motion. Further investigations demonstrated that a cluster of arginines in the C-terminus of MLKL is important for the molecular conformational switch. Functional mutagenesis showed that mutating these arginines to glutamates hindered the membrane disruption of full-length MLKL and thus inhibited the necroptotic cell death. These findings suggest that the brace helix also plays an active role in MLKL regulation, rather than an auto-inhibitory domain.
细胞程序性坏死是一种通过混合谱系激酶结构域样蛋白（MLKL）导致细胞膜破坏的细胞死亡方式。先前的研究表明，MLKL 的 N 端四螺旋束结构域（NBD）是导致膜通透性的执行结构域，该结构域被第一个支撑螺旋（H6）所抑制。在细胞程序性坏死发生后，这个抑制性的支撑螺旋会脱落，NBD 可以整合到细胞膜中，从而导致细胞程序性坏死。然而，NBD 是如何释放并诱导膜破裂的，目前还不清楚。在这里，我们将 MLKL 重组到模拟细胞膜的双体脂中，并观察到第一个支撑螺旋的结构破坏和膜释放，这是由带负电荷的磷脂以剂量依赖的方式调节的。通过分子动力学模拟，我们发现，在 7 ns 的动态运动后，孤立的、自我抑制的 MLKL 中的支撑区在溶液中会变得固有无序。进一步的研究表明，MLKL 末端的精氨酸簇对于分子构象转换很重要。功能突变表明，将这些精氨酸突变为谷氨酸会阻碍全长 MLKL 的膜破坏，从而抑制细胞程序性坏死。这些发现表明，支撑螺旋在 MLKL 的调节中也发挥着积极的作用，而不仅仅是作为一个自我抑制的结构域。
