Necroptotic cell death is mediated by the most terminal known effector of the pathway, MLKL. Precisely how phosphorylation of the MLKL pseudokinase domain activation loop by the upstream kinase, RIPK3, induces unmasking of the N-terminal executioner four-helix bundle (4HB) domain of MLKL, higher-order assemblies, and permeabilization of plasma membranes remains poorly understood. Here, we reveal the existence of a basal monomeric MLKL conformer present in human cells prior to exposure to a necroptotic stimulus. Following activation, toggling within the MLKL pseudokinase domain promotes 4HB domain disengagement from the pseudokinase domain αC helix and pseudocatalytic loop, to enable formation of a necroptosis-inducing tetramer. In contrast to mouse MLKL, substitution of RIPK3 substrate sites in the human MLKL pseudokinase domain completely abrogated necroptotic signaling. Therefore, while the pseudokinase domains of mouse and human MLKL function as molecular switches to control MLKL activation, the underlying mechanism differs between species.

中文翻译：

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假激酶结构域的构象转换促进人MLKL四聚化和坏死性细胞死亡。

坏死性细胞死亡是由该途径的最末端已知的效应子MLKL介导的。确切地讲，上游激酶RIPK3对MLKL假激酶结构域激活环的磷酸化如何诱导MLKL的N末端执行者四螺旋束（4HB）域的解蔽，更高阶的组装以及质膜的通透性仍然知之甚少。在这里，我们揭示了暴露于坏死性刺激之前人类细胞中存在的基础单体MLKL构象体的存在。激活后，在MLKL假激酶结构域内切换可促进4HB结构域从假激酶结构域αC螺旋和假催化环上脱离，从而能够形成诱导坏死病的四聚体。与鼠标MLKL相比，MLKL假激酶结构域中RIPK3底物位点的取代完全废除了坏死性信号传导。因此，虽然小鼠和人MLKL的假激酶结构域起着控制MLKL激活的分子开关的作用，但不同物种之间的潜在机制却有所不同。