Multiple domain interfaces mediate SARM1 autoinhibition

Abstract

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.

Keywords: NMN; axonopathy; metabolism; neurodegeneration; neuropathy.

Fig. 1.

A conserved hydrophobic region of peptide 5 mediates inhibition of SARM1 NADase and prodegenerative activity. (A) Domain organization of SARM1 and the enzymatic reaction that it catalyzes. SARM1 can hydrolyze NAD⁺ into either ADPR and nicotinamide (Nam) or cyclic ADPR (cADPR) and Nam. (B) Schematic of the N-terminal region encompassed by peptide 5 and sequences of peptides derived from peptide 5. Black letters correspond to hydrophobic residues, green letters to nonpolar residues, magenta letters to polar residues, blue letters to basic residues, and red letters to acidic residues. The size of the residue letter indicates the frequency of that amino acid(s) at that position. (C) Twofold dose–response curves for peptide-mediated inhibition of SARM1:SAM-TIR NADase activity in vitro. Error bars represent ± SEM. n = 4 to 6; two-way ANOVA was used for statistical analysis with ***P < 0.001 with asterisk color denoting a concentration of a given peptide having an effect significantly different from measurements without the peptide. (D) DRG sensory neurons from SARM1 KO mice were infected with variants of SARM1, and metabolites were extracted 3 dpi to measure cADPR levels relative to neurons infected with wild-type SARM1. Error bars represent SEM; n = 4; one-way ANOVA with Dunnett posttest was used for statistical analysis with *P < 0.05 and **P < 0.01. (E) DRG sensory neurons from SARM1 KO mice were infected with variants of SARM1, and axons were imaged 5 d later. Axon fragmentation is quantified using the axon degeneration index (29). Error bars represent SEM, n = 4 to 5; one-way ANOVA with Dunnett posttest was used for statistical analysis with ***P < 0.001 and n.s. meaning not significantly different from control.

Fig. 2.

The M5 region inhibits SARM1 activation in cells. (A) Wild-type SARM1 with Cerulean attached to the N terminus and Venus attached to the C terminus exhibited a positive FRET/donor ratio in HEK293T cells, but similarly tagged SARM1:M5 did not. Error bars represent SEM; n = 4; one-way ANOVA with Bonferroni posttest was used for statistical analysis with **P < 0.01 and ***P < 0.001. (B) DRG sensory neurons from SARM1 KO mice were infected with SARM1:SAM-TIR mutant along with constructs expressing concatemers of either the wild-type peptide 5 region or the peptide 5 M5 mutant region. Metabolites were extracted at 3 d after infection to measure cADPR levels. Metabolite levels are shown as compared to those observed in neurons infected with wild-type SARM1 alone. Error bars represent SEM; n = 4; one-way ANOVA with Dunnett posttest was used for statistical analysis with *P < 0.05 and **P < 0.01. (C) DRG sensory neurons from SARM KO mice were imaged at 5 dpi to measure axon degeneration. Error bars represent SEM; n = 4; one-way ANOVA with Dunnett posttest was used for statistical analysis with ***P < 0.001 and n.s. meaning not significantly different from control.

Fig. 3.

Overall structure of human SARM1. (A) Domain arrangement of SARM1 and the octameric model fitted into the composite EM map combining the C8 symmetry map and the TIR-focused refined C1 map. The dimensions of the octamer are indicated. (B) Ribbon diagram of the octameric model, color coded by domains. ARM: light pink; SAM: sky blue; TIR: wheat. (C) Five intramolecular and intermolecular interfaces between SARM1 domains are shown on two extracted protomers.

Fig. 4.

Structural and functional analyses of the ARM-TIR interfaces in the octamer. (A) Interactions involved in ARM-TIR interface I (Left) and ARM-TIR interface II (Right). (B and C) Functional effects of mutations at these two interfaces. DRG sensory neurons from SARM1 KO mice were infected with various SARM1 constructs with indicated mutations in ARM-TIR interface I (B) or ARM-TIR interface II (C). Metabolites were extracted 3 dpi to measure cADPR levels relative to neurons infected with wild-type SARM1. Error bars represent SEM; n = 3 to 4. One-way ANOVA with Dunnett posttest was used for statistical analysis with *P < 0.05 and ***P < 0.001.

Fig. 5.

Structural and functional analyses of the ARM-ARM and ARM-SAM interfaces. (A) Interactions involved in the ARM-ARM interface. (B) Functional effects of mutations at the ARM-ARM interface. DRG sensory neurons from SARM1 KO mice were infected with SARM1 constructs with mutations in the ARM-ARM interface. Metabolites were extracted 3 dpi to measure cADPR levels relative to neurons infected with wild-type SARM1. (C) Interactions involved in ARM-SAM interface I (Left) and ARM-SAM interface II (Right). (D and E) Functional effects of mutations at ARM-SAM interfaces. DRG sensory neurons from SARM1 KO mice were infected with SARM1 constructs with mutations in ARM-SAM interface I (D) or ARM-SAM interface II (E). Metabolites were extracted 3 dpi to measure cADPR levels relative to neurons infected with wild-type SARM1. Error bars represent SEM; n = 3 to 4; one-way ANOVA with Dunnett posttest was used for statistical analysis with **P < 0.01 and ***P < 0.001.