Stony Brook University Cancer Center, Stony Brook, NY 11794.
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794.
Proc Natl Acad Sci U S A. 2017 Jul 11;114(28):E5549-E5558. doi: 10.1073/pnas.1705134114. Epub 2017 Jun 26.
Neutral sphingomyelinase 2 (nSMase2, product of the gene) is a key enzyme for ceramide generation that is involved in regulating cellular stress responses and exosome-mediated intercellular communication. nSMase2 is activated by diverse stimuli, including the anionic phospholipid phosphatidylserine. Phosphatidylserine binds to an integral-membrane N-terminal domain (NTD); however, how the NTD activates the C-terminal catalytic domain is unclear. Here, we identify the complete catalytic domain of nSMase2, which was misannotated because of a large insertion. We find the soluble catalytic domain interacts directly with the membrane-associated NTD, which serves as both a membrane anchor and an allosteric activator. The juxtamembrane region, which links the NTD and the catalytic domain, is necessary and sufficient for activation. Furthermore, we provide a mechanistic basis for this phenomenon using the crystal structure of the human nSMase2 catalytic domain determined at 1.85-Å resolution. The structure reveals a DNase-I-type fold with a hydrophobic track leading to the active site that is blocked by an evolutionarily conserved motif which we term the "DK switch." Structural analysis of nSMase2 and the extended N-SMase family shows that the DK switch can adopt different conformations to reposition a universally conserved Asp (D) residue involved in catalysis. Mutation of this Asp residue in nSMase2 disrupts catalysis, allosteric activation, stimulation by phosphatidylserine, and pharmacological inhibition by the lipid-competitive inhibitor GW4869. Taken together, these results demonstrate that the DK switch regulates ceramide generation by nSMase2 and is governed by an allosteric interdomain interaction at the membrane interface.
中性鞘磷脂酶 2（nSMase2，基因产物）是一种关键的神经酰胺生成酶，参与调节细胞应激反应和细胞间外泌体介导的通讯。nSMase2 被多种刺激物激活，包括阴离子磷脂酰丝氨酸。磷脂酰丝氨酸与完整膜的 N 端结构域（NTD）结合；然而，NTD 如何激活 C 端催化结构域尚不清楚。在这里，我们确定了 nSMase2 的完整催化结构域，由于插入较大而被错误注释。我们发现可溶性催化结构域与膜相关的 NTD 直接相互作用，NTD 既是膜锚定物又是别构激活物。连接 NTD 和催化结构域的跨膜区是激活所必需的且是充分的。此外，我们使用分辨率为 1.85 Å 的人 nSMase2 催化结构域的晶体结构提供了这种现象的机制基础。该结构揭示了一种 DNase-I 型折叠，其中存在一条通向活性位点的疏水区，该区域被一个保守的基序（我们称之为“DK 开关”）阻塞。对 nSMase2 和扩展的 N-SMase 家族的结构分析表明，DK 开关可以采用不同的构象来重新定位参与催化的普遍保守的天冬氨酸（D）残基。nSMase2 中该天冬氨酸残基的突变破坏了催化、别构激活、磷脂酰丝氨酸刺激以及脂质竞争性抑制剂 GW4869 的药理学抑制作用。总之，这些结果表明，DK 开关通过 nSMase2 调节神经酰胺的生成，并受膜界面上的别构域间相互作用控制。
