State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, 518055, China.
Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China.
Nat Commun. 2022 Dec 22;13(1):7898. doi: 10.1038/s41467-022-35581-y.
Sterile alpha (SAM) and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1) is an autoinhibitory NAD-consuming enzyme that is activated by the accumulation of nicotinamide mononucleotide (NMN) during axonal injury. Its activation mechanism is not fully understood. Here, we generate a nanobody, Nb-C6, that specifically recognizes NMN-activated SARM1. Nb-C6 stains only the activated SARM1 in cells stimulated with CZ-48, a permeant mimetic of NMN, and partially activates SARM1 in vitro and in cells. Cryo-EM of NMN/SARM1/Nb-C6 complex shows an octameric structure with ARM domains bending significantly inward and swinging out together with TIR domains. Nb-C6 binds to SAM domain of the activated SARM1 and stabilized its ARM domain. Mass spectrometry analyses indicate that the activated SARM1 in solution is highly dynamic and that the neighboring TIRs form transient dimers via the surface close to one BB loop. We show that Nb-C6 is a valuable tool for studies of SARM1 activation.
无菌α（SAM）和 Toll/白细胞介素-1 受体（TIR）结构域包含 1（SARM1）是一种自动抑制 NAD 消耗酶，在轴突损伤过程中会被烟酰胺单核苷酸（NMN）的积累所激活。其激活机制尚不完全清楚。在这里，我们生成了一种纳米抗体 Nb-C6，它特异性识别 NMN 激活的 SARM1。Nb-C6 仅在使用穿透性 NMN 模拟物 CZ-48 刺激的细胞中标记激活的 SARM1，并在体外和细胞中部分激活 SARM1。NMN/SARM1/Nb-C6 复合物的冷冻电镜显示出一个八聚体结构，ARM 结构域明显向内弯曲，并与 TIR 结构域一起摆动。Nb-C6 结合到激活的 SARM1 的 SAM 结构域，并稳定其 ARM 结构域。质谱分析表明，溶液中的激活的 SARM1 非常不稳定，相邻的 TIR 通过接近一个 BB 环的表面形成短暂的二聚体。我们表明，Nb-C6 是研究 SARM1 激活的有价值的工具。
