Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Stepwise activation of SARM1 for cell death and axon degeneration revealed by a biosynthetic NMN mimic.

doi: 10.1073/pnas.2424906122

Figure Lengend Snippet: Fig. 5. Introducing a disulfide bond between the ARM and SAM domains inhibited M1 or NMN- mediated SARM1 activation. (A) Cell survival rates in HeLa-SARM1−/− cells (Vector) and HeLa-SARM1−/− cells ectopically expressing SARM1 or SARM1 cysteine mutants, treated with 10 μM G10 for 24 h, determined by measuring ATP levels. Data are represented as mean ± SD from triplicate wells. (B) Time course of the base exchange activities of the purified SARM1 (WT) or SARM1 (2Cmut) incubated with DPBS or M1. Each bar represents mean ± SD, n = 3. ****P < 0.0001, n.s., not significant. (C and D) Relative NAD+ (C) and cADPR (D) levels in the reaction mixture containing purified SARM1 WT (Top) or 2Cmut (Bottom) incubated with increasing concentrations of NMN or M1 for 30 min. Each bar represents mean ± SD, n = 3. (E) Cryo-EM map of SARM1 2Cmut in complex with M1. The model is fitted in the map. (F) Comparison of structures of SARM1 WT (colored in yellow) and 2Cmut (colored by domains) in complex with M1. (G) Close-up view of the secondary binding interface. A portion of the BB loop (e.g., D605 and E604) in the structure of 2Cmut is resolved through interactions with the ARM domain (e.g., K173). (H) EM density of the disulfide bond in the 2Cmut at a contour level of 4σ.

Article Snippet: Similarly, 0.1 μg/μL SARM1 (WT) was incubated with 500 μM DSARM (MCE) and 500 μM NAD+ (MCE) in DPBS buffer containing an additional 150 mM NaCl and rotated at room temperature overnight.

Techniques: Activation Assay, Plasmid Preparation, Expressing, Purification, Incubation, Cryo-EM Sample Prep, Comparison, Binding Assay