Axon degeneration is an active program of self-destruction mediated by the protein SARM1. In healthy neurons, SARM1 is autoinhibited and, upon injury autoinhibition is relieved, activating the SARM1 enzyme to deplete NAD⁺ and induce axon degeneration. SARM1 forms a homomultimeric octamer with each monomer composed of an N-terminal autoinhibitory ARM domain, tandem SAM domains that mediate multimerization, and a C-terminal TIR domain encoding the NADase enzyme. Here we discovered multiple intramolecular and intermolecular domain interfaces required for SARM1 autoinhibition using peptide mapping and cryo-electron microscopy (cryo-EM). We identified a candidate autoinhibitory region by screening a panel of peptides derived from the SARM1 ARM domain, identifying a peptide mediating high-affinity inhibition of the SARM1 NADase. Mutation of residues in full-length SARM1 within the region encompassed by the peptide led to loss of autoinhibition, rendering SARM1 constitutively active and inducing spontaneous NAD⁺ and axon loss. The cryo-EM structure of SARM1 revealed 1) a compact autoinhibited SARM1 octamer in which the TIR domains are isolated and prevented from oligomerization and enzymatic activation and 2) multiple candidate autoinhibitory interfaces among the domains. Mutational analysis demonstrated that five distinct interfaces are required for autoinhibition, including intramolecular and intermolecular ARM-SAM interfaces, an intermolecular ARM-ARM interface, and two ARM-TIR interfaces formed between a single TIR and two distinct ARM domains. These autoinhibitory regions are not redundant, as point mutants in each led to constitutively active SARM1. These studies define the structural basis for SARM1 autoinhibition and may enable the development of SARM1 inhibitors that stabilize the autoinhibited state.

轴突变性是由蛋白质 SARM1 介导的主动自我毁灭程序。在健康的神经元中，SARM1 被自身抑制，并且在损伤后自身抑制被解除，激活 SARM1 酶以消耗 NAD ⁺并诱导轴突变性。SARM1 形成同型多聚体八聚体，每个单体由 N 端自抑制 ARM 结构域、介导多聚化的串联 SAM 结构域和编码 NADase 酶的 C 端 TIR 结构域组成。在这里，我们使用肽图谱和低温电子显微镜 (cryo-EM) 发现了 SARM1 自身抑制所需的多个分子内和分子间域界面。我们通过筛选一组源自 SARM1 ARM 结构域的肽，确定了一个候选的自身抑制区域，确定了一种介导 SARM1 NADase 高亲和力抑制的肽。肽所包含区域内全长 SARM1 残基的突变导致自身抑制的丧失，使 SARM1 具有组成型活性并诱导自发的 NAD ⁺和轴突损失。SARM1 的低温 EM 结构揭示了 1) 一个紧凑的自抑制 SARM1 八聚体，其中 TIR 结构域被隔离并防止寡聚化和酶促活化；2) 结构域之间存在多个候选自抑制界面。突变分析表明，自抑制需要五个不同的界面，包括分子内和分子间 ARM-SAM 界面、分子间 ARM-ARM 界面以及在单个 TIR 和两个不同 ARM 结构域之间形成的两个 ARM-TI​​R 界面。这些自抑制区域不是多余的，因为每个点突变体都导致了组成性活跃的 SARM1。这些研究确定了 SARM1 自身抑制的结构基础，并可能有助于开发稳定自身抑制状态的 SARM1 抑制剂。