Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, 3010, Australia.
Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia.
Cell Death Differ. 2022 Sep;29(9):1804-1815. doi: 10.1038/s41418-022-00965-6. Epub 2022 Mar 9.
Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL's N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.
细胞程序性坏死是一种先天免疫起源的溶细胞性细胞死亡途径，在炎症性疾病中经常失调。该途径的终末效应子 MLKL 在其上游调节因子 RIPK3 激酶磷酸化其假激酶结构域后被许可杀伤。磷酸化促使 MLKL 的 N 端四螺旋束（4HB 或 HeLo）结构域释放，该结构域结合并透化质膜导致细胞死亡。4HB 结构域透化质膜的确切机制以及该机制在不同物种之间的差异尚不清楚。在这里，我们使用 NMR 光谱鉴定了小鼠 MLKL 的膜结合表位。通过脂质体透化和细胞死亡测定，我们验证了α3 螺旋中的 K69、α4 螺旋中的 W108 以及第一臂螺旋中的 R137/Q138 是坏死信号的关键残基。该表位与报道的人 MLKL 的磷脂结合位点不同，人 MLKL 的结合位点包含主要位于α1 和α2 螺旋中的碱性残基。与人和植物 MLKL 同源物进一步形成对比的是，α3-α4 环在溶液中形成螺旋，而在小鼠 MLKL 中该环未形成结构。这些发现共同说明了 4HB 结构域折叠的多功能性，其溶细胞功能可以由不同同源物中的不同表位介导。
