The Structural Basis for SARM1 Inhibition, and Activation Under Energetic Stress

SARM1 is a central executor of axonal degeneration (1). Mechanistically, SARM1 contains NADase activity, which, in response to nerve injury, depletes the key cellular metabolite, NAD+ (2–5). Interestingly, SARM1 knockout mouse models do not present any apparent physiological impairment. Yet, the lack of SARM1 protects against various neuropathies (6, 7), thereby highlighting SARM1 as a likely safe and effective drug target (8). However, the absence of a SARM1 structure, in its active or inhibited form, makes it impossible to understand the molecular basis of SARM1 inhibition, and its activation under stress conditions. In this study we present two cryo-EM maps of SARM1 (at 2.6 Å and 2.9 Å resolution). We show that the inhibited SARM1 homo-octamer assumes a packed conformation with well-ordered inner and peripheral rings. Here the catalytic TIR domains are held apart from each other and are unable to form dimers, which is a prerequisite for NADase activity. More importantly, after screening several cellular metabolites we discovered that the inactive conformation is stabilized by the binding of SARM1’s own substrate: NAD+. The NAD+ inhibitory allosteric site is located away from the NAD+ catalytic site of the TIR domain. Site-directed mutagenesis of the allosteric site leads to constitutive active SARM1. Based on our data we propose that a reduction of cellular NAD+ concentrations (an early indication of disease-associated and age-related neurodegeneration (9)) disassemble SARM1’s peripheral ring, which allows NADase activity. This leads to an energetic catastrophe and eventually cell death. The discovery of the allosteric inhibitory site opens the door for the development of effective drugs that will prevent SARM1 activation, rather than compete for binding to the NADase catalytic site. Brief description It is not known how NAD+ depletion brings about neurodegeneration. Here, we show that the intrinsic NADase activity of SARM1 is allosterically inhibited by physiological concentrations of NAD+. NAD+ stabilizes a compact, auto-inhibited conformation of the SARM1 octamer. Once NAD+ levels are depleted, the allosteric inhibition is released, enabling SARM1’s NADase activity, which eventually leads to energetic catastrophe and cell death.

Introduction SARM1 (sterile α and HEAT/armadillo motif-containing protein (10)) was first discovered as a negative regulator of TRIF (TIR domain-containing adaptor inducing interferon-β) in TLR (Tolllike receptor) signaling (11), and was later shown to promote neuronal death by oxygen and glucose deprivation (7) and viral infections (6,(12)(13)(14), while also having a protective role against bacterial and fungal infections in C. elegans (15,16). Multiple studies have demonstrated that SARM1 is a key part of a highly conserved axonal death pathway that is activated by nerve injury (17,18). Importantly, recent studies have shown that SARM1 deficiency confers protection against axonal degeneration in several models of neurodegenerative conditions (6,7,19,20), making SARM1 a compelling molecular target for the development of safe and effective pharmacological therapy to protect axons in a variety of axonopathies (8).
Amino acid substitution (E642A) at the TIR domain's active site (3) abolishes the NADase activity in vitro and inactivates SARM1 pro-degenerative activity (4,5), thereby linking the role of SARM1 in axonal degeneration with its NADase activity. The enzymatic activity requires a high local concentration of the TIR domains, as demonstrated by forced dimerization of TIR, which resulted in NAD+ hydrolysis and neuronal cell death (2,24). Also, in the absence of the auto-inhibitory ARM domain, which can interact directly with TIR (22), the remaining SAM-TIR construct is active and leads to rapid cell death (18,25). The mechanism by which SAM domains cause TIR crowding became clearer in our (25) recent report, where we showed that both hSARM1 and the isolated tandem SAM1-2 domains form octamers in solution. Also, we used negative stain electron microscopy analysis of hSARM1 and determined the crystal structure (as did others (3)) of the SAM1-2 domains -both of which revealed an octameric ring arrangement.
Based on these findings, it appears that hSARM1 is kept auto-inhibited by the ARM domain in homeostasis, and gains NADase activity upon the infliction of injury (axotomy (17)), oxidative (mitochondria depolarization (26); oxidizing agents (27)), metabolic (depletion of NAD+ (28)), or toxic (chemotherapy drugs (29)) stress conditions. Whether and how all or some of these insults converge to induce SARM1 activation is still not completely understood. In this regard, little is known about the direct molecular triggers of SARM1 in cells, besides the potential involvement of nicotinamide mononucleotide (NMN) (30,31) and Ser-548 phosphorylation by JNK (32) in promoting the NADase activity of SARM1. Here we present structural data and complementary biochemical assays, to show that SARM1 is kept inactive through a 'substrate inhibition' mechanism, where high concentration of NAD+ stabilizes the tightly packed, inhibited conformation of the protein. In this way, SARM1 activation is triggered by a decrease in the concentration of a cellular metabolite -NAD+, rather than by the introduction of an activating factor.

Cryo-EM visualization of purified hSARM1
For cryo-EM imaging, the near-intact hSARM1, short of the N' terminal mitochondrial localization signal (26ERL…GPT724) and mutated in the NADase catalytic residue E642Q, was expressed in mammalian cell culture and isolated to homogeneity using consecutive metal chelate and size exclusion chromatography. hSARM1w.t. (26ERL…GPT724) that was not mutated in E642, was also expressed and isolated (Fig. 1B), although with lower yields, and was used in cellular and in vitro activity assays. We first collected cryo-EM images of the purified hSARM1E642Q. 2D classification ( Fig. 1C) and 3D reconstruction (Fig. 1D) revealed an octamer ring assembly with clear depiction of the inner ring, which is attributed to the tandem SAM domains. Only a minor fraction of the particles (~20%) shows the presence of a partial peripheral ring composed by the ARM and TIR domains. Cryo-EM analysis of the isolated SAM1-2 domains (Fig. 1D), and docking of the crystal structure of the SAM1-2 domains' octamer ring (PDB code 6QWV) into the 3D maps demonstrates that indeed, the ARM and TIR domains are largely missing from this reconstruction, implying a disordered outer ring in ~80% of the particles. Exploring different buffers, pH and salt conditions, addition of various detergents, as well as variations in cryo-EM grid preparation (e.g. ice thickness) did not affect the visibility of the octamer outer ring considerably.
These results are inconsistent with our previous analysis, where we used low resolution negative stain EM visualization and 2D classification of hSARM1E642Q that showed fully assembled inner and outer ring structures (Fig. 1C) (25). We consider, that a gradient fixation (GraFix (33)) step that involves ultra-centrifugation of the protein sample through a glycerol + glutaraldehyde crosslinker gradients, which was applied before the negative stain -but not before the cryo-EM sample preparations -might be the cause for the difference between the two measurements. We therefore pursued cryo-EM data collection of GraFix-ed hSARM1E642Q after dilution of the glycerol from 18% (which severely diminishes protein contrast in cryo-EM) to 2.5%.

2.8-6.5 Å resolution structure of a fully assembled compact hSARM1 GraFix-ed octamer
We carried out 2D classification ( Fig. 2A) and 3D reconstruction and refinement ( Fig. 2B-D) of the GraFix-ed hSARM1E642Q to 2.8-6.5 Å resolution (applying 8-fold symmetry). Overall, the hSARM1E642Q octamer is 203 Å in diameter and 80 Å thick (Fig. S2A). The SAM1-2 domains' inner ring is the best resolved part of the map, to which the high-resolution crystal structure (PDB code 6QWV) was fitted with minute adjustments. The TIR domains are the least defined part of the density map, mostly not revealing side chain positions (Fig. S3A). However, the availability of high-resolution crystal structures of isolated hSARM1 TIR (PDB codes 6O0R, 6O0U) allowed their docking into the well resolved secondary structure elements in the map with very high confidence. The ARM domains show intermediate quality, with well resolved secondary structures and bulky sidechains. This allowed the building of a de-novo atomic model for the entire ARM (Fig. S2A,B), as no high-resolution structure or homology models of this part of SARM1 are available. The entire atomic model comprises residues 56-700 (Fig. S1), with an internal break at the linker, which connects SAM2 to the TIR domain. The structural analyses of the SAM domains and the SAM octamer ring assembly, as well as the atomic details of the TIR domain, are described in our previous study, and by others (3,25). The cryo-EM structure reveals a closed crescent-shaped ARM region, composed of seven three-helix ARM repeats spanning residues 60-400 (Fig. S2B). The ARM topology is split into two interacting parts, designated ARM1 (res. 60-303 with five ARM repeats) and ARM2 (res. 322-400 with two ARM repeats) (Fig.   S1, S2B). The main ARM1 -ARM2 interaction interface is hydrophobic and involves helices 14 and 16 of ARM1 and helices 1, 2 and 3 of ARM2. ARM1 and ARM2 are also interacting at the crescent 'horns' through the ARM1 2-3 loop with the loop that connects ARM1 with ARM2 (res. 305-320). In the hSARM1 compact octamer, each ARM is directly connected via a linker (res. 400-404) to a SAM1 domain. Also, each ARM is engaged in several non-covalent interactions with the same-chain SAM and with the clockwise neighboring SAM, when assuming a top view of the structure (Fig. 2B,D). Although neighboring ARM domains are closely packed, direct interactions between them seem to be limited, engaging a short segment of ARM1 9 with the 4-5 loop of ARM2 of the neighboring chain. Additional ARM-ARM interactions are indirect, mediated by the TIR domains. Each TIR binds the ARM ring via two sites, designated the 'primary' and 'secondary' TIR docking sites (Fig. 3A). The 'primary' is larger and engages the TIR helix A and the EE loop with the ARM1 10, 10-11 loop and 13. The 'secondary' TIR docking site is smaller and involves the TIR BB loop and helix 7 of the counter-clockwise ARM1 domain ( Fig. S1).

The compact conformation of hSARM1 is inhibited for NADase activity
The GraFix-ed cryo-EM structure revealed a domain organization where the catalytic TIR subunits are separated from each other by their docking onto the ARM peripheral ring, the assembly of which is dependent on the coupling of each TIR with two neighboring ARM domains ( Fig. 3A). Since the NADase activity of TIR requires homo-dimerization (2,24), we considered that the cryo-EM structure represents an inhibited conformation of SARM1, in which the TIR domains cannot form compact dimers and catabolize NAD+. To test this assumption, we designed amino acid substitutions at the ARM's primary TIR docking site, aimed to weaken TIR docking and thus allow their dimerization and subsequent NAD+ catalysis, but without compromising the protein's structural integrity -particularly that of the TIR domain (Fig. 3A). Two pairs of mutations -RR216-7EE of ARM1 helix 10, and FP255-6RR of ARM1 helix 13 (Fig. 3C, S1) were introduced, as well as the double mutant RR216-7EE/FP255-6RR, and transiently expressed in HEK-293T cells. Their expression effects on NAD+ levels and cell viability were monitored using a previously demonstrated method, the resazurin fluorescence assay (18,34).
The results (Fig. 3C) show a rapid decrease in cellular NAD+ levels and 50% cell death 24 hours after transfection of the FP255-6RR and double mutant. This toxicity level is similar to that of a hSARM1 construct missing the entire ARM domain 'delARM' (res. 409-724) (Fig. 3C), which was previously shown to be toxic in neurons and HEK293 cells (18,25). This toxic effect was attributed to the removal of auto-inhibitory constraints imposed by the ARM domain. The RR216-7EE mutation has a weaker effect, probably due to the position of these amino acid residues at the margin of the TIR-ARM interface.
In conclusion, we found that hSARM1 inhibition requires ARM-TIR interaction through the 'primary TIR docking site'. We further calculated the surface conservation scores of SARM1 orthologs. The scores were color coded and plotted on the molecular surface of hSARM1 using the Consurf server ( Fig. 3B) (35). The surface-exposed residues reveal a high level of conservation on both the ARM and TIR domains at the binding interface, highlighting the biological importance of the interaction and the possible conservation of its function in autoinhibition. It was previously suggested, that in order to maintain auto-inhibition, SARM1 is kept as a monomer, that upon activation, undergoes multimeric assembly (1), very much like other apoptotic complexes. Our results show otherwise, and explain how hSARM1 avoids premature activation even as a pre-formed octamer, which may allow rapid activation and response.

Isolated hSARM1 is NADase active in vitro and inhibited by glycerol
As it became clear that the compact two-ring structure is inhibited for NADase activity, we considered whether the purified hSARM1, that was not subjected to GraFix, and predominantly presents just the inner SAM ring in cryo-EM 2D averaging and 3D reconstruction (Fig. 1C,D), is active in vitro. Using a resazurin fluorescence assay modified for in vitro application, we measured the rate of NAD+ consumption by hSARM1w.t. in a series of NAD+ concentrations and determined a Km of 28±4 µM, with Vmax of 9±0.3 µM/min (Fig. 3D). This rate was remarkably similar to the previous kinetic study of SARM1, which reported a Km of 24 µM (4) -taking into account, that the previous study used an isolated TIR domain fused to artificial dimerizing and aggregating agents, while we used the near-intact protein.
It was previously demonstrated that nicotinamide mononucleotide (NMN) and a membranepermeable NMN analogue could activate SARM1 in cultured cells (30,31). We therefore measured the effect of NMN supplement on the NADase activity of purified hSARM1 and found a moderate 30% increase in activity with 1mM NMN (Fig. 3E). These results demonstrate that the purified hSARM1 is indeed already mostly NADase active, even without NMN supplement, consistent with the predominant open conformation seen in cryo-EM (Fig. 1C,D).
Next, we examined what confines the GraFix-ed hSARM1 into the compact inhibited conformation (Fig. 2), and measured the in vitro NADase activity in the presence of glycerol (Fig.   3F). We found that glycerol reduces hSARM1 NADase activity in a concentration-dependent manner, reaching 72% inhibition at 15% glycerol. In the GraFix preparation, we extracted hSARM1 from the gradient tube after it reached approximately 18% glycerol concentration, where SARM1 is at the inhibited compact conformation. It seems likely, that this conformation is maintained after glycerol is removed, due to glutaraldehyde crosslinking which preserves the compact structure.

NAD+ substrate inhibition of hSARM1
Our results show that hSARM1 NADase activity is suppressed in cell culture, but much less so in vitro after being isolated. Our hypothesis was, that in the course of purification from the cytosolic fraction, hSARM1 loses a low-affinity cellular factor, responsible for inhibiting it in the cellular environment. Since SARM1 was previously shown to be activated in cell culture in response to metabolic, toxic and oxidative stresses, we considered that, perhaps, the hypothesized inhibitory co-factor is a small molecule that is depleted under cellular stress conditions and thus, the inhibition of hSARM1 is released. To follow through on this hypothesis, we tested the impact of several small molecules, which are associated with the cell's energetic state, with two in vitro parameters: hSARM1 NADase activity, and structural conformation (visualized by cryo-EM). We have already established that glycerol meets these two criteria, as it imposes hSARM1 compact conformation ( Fig. 2) and reduces NADase activity (Fig. 3F). Glycerol was also found to occupy the active site of the hSARM1 TIR domain in a crystal structure (PDB code 6O0R), directly linking structural data with enzymatic inhibition. However, the glycerol concentrations in which these in vitro experiments were conducted were very high (15-18% for the NADase activity assay and cryo-EM, and 25-30% in the X-ray crystal structure), i.e. 2-4 M, which is considerably higher than the estimated sub 1mM concentrations of intracellular physiological glycerol (36). We next considered that ATP, being the main energetic compound, with cellular concentrations of 1-10mM, which are depleted in cell death and axonal degeneration, may alternatively serve as a fit candidate to inhibit hSARM1. Indeed, we found that ATP inhibits the NADase activity of hSARM1 in a dose-dependent manner (Fig. 4A). However, it did not affect hSARM1 conformation as observed by 2D classification (Fig. 4B). Therefore, it is possible that ATP is a competitive inhibitor of hSARM1, which bind to the TIR domain's active site, and the cryo-EM data further suggest, that a different, allosteric site, is affecting the major changes observed in hSARM1 conformation.
It was previously shown that ablation of the cytosolic NAD+ synthesizing enzymes NMNAT1 and 2 decreases cytoplasmic NAD+, and induces SARM1 activation (28,37). Therefore, we postulated that it is the high physiologic concentration of NAD+ itself, that may inhibit hSARM1 through 'substrate inhibition' -a general mechanism regulating the activity of many enzymes (38).
In cryo-EM visualization, the effect of adding 5-mM NAD+ to hSARM1 was dramatic, showing >80% of the particles in the two-ring, compact conformation -in contrast to the <20% shown in the absence of NAD+ (Fig. 4B,C). To measure NAD+ substrate inhibition of hSARM1, we applied a fluorescent assay using a wide concentrations range of the NAD+ analog, etheno-NAD (eNAD) (mixed 1:10 mol/mol with regular NAD+) that fluoresces upon hydrolysis (ex. λ = 330 nm, em. λ = 405 nm). The results showed a bell-shape curve ( Fig. 4D) with the highest rate of hydrolysis at 100µM NAD+ and a steady decrease of activity thereafter, in the higher concentrations. In addition, we used a direct reverse-phase HPLC monitoring of NAD+ consumption by hSARM1 compared with porcine brain NADase. While the rate of hydrolysis was maintained between 50µM and 2mM NAD+ by the porcine NADase, hSARM1 was clearly inhibited by the 2mM NAD+ ( Fig. 4E).

Å resolution structure of NAD+ induced hSARM1 compact octamer
Following the cryo-EM 2D classification ( Fig. 4B,C) and enzymatic (Fig. 4D,E) indications for NAD+ substrate inhibition of hSARM1, we pursued a 3D structure determination of hSARM1 complexed with NAD+ at inhibiting concentrations. For this purpose, 3D reconstruction of hSARM1 supplemented by 5mM NAD+ was performed after particle picking and 2D classification ( Fig. 5A), and was refined to 2.7 Å resolution, with excellent map quality ( Fig. 5 and Fig. S4). The structure was largely identical to the GraFix-ed structure, further substantiating the validity of the latter (Fig. 5B). Two small structural differences can be observed between the NAD+ supplemented density map and that of the GraFix-ed hSARM1. The first difference is a 5Å shift in the position of the distal part of the TIR domain, and the second is a rearrangement of the secondary structure at the region of the 'crescent horns', where the tips of ARM1 and ARM2 touch ( Fig. 5C, S4A). In the same region, designated hereafter 'ARM allosteric site', an extra-density in the NAD+ supplemented map reveals the binding site of one NAD+ molecule, providing clear atomic details (Fig. 5C, right panel). To probe into the function of the ARM allosteric site, we introduced point mutations, targeting three structural elements surrounding the NAD+ density.
These elements were: ARM1 2; ARM1 5-6 loop; and the ARM1-ARM2 loop (Fig. 5C,D). For control, we introduced two other mutations in sites that we do not consider to be involved in hSARM1 inhibition or activation. We hypothesized that NAD+ binding at the ARM allosteric site stabilizes the ARM conformation by interacting with both ARM1 and ARM2, thereby promoting the hSARM1 compact auto-inhibited structure. Therefore, mutations in the allosteric site, that interfere with NAD+ binding, would diminish auto-inhibition and allow hSARM1 activity in cell culture. Indeed, we found that two separate mutations in the ARM1 5-6 loop (L152A, and R157E) and one in the opposite ARM1-ARM2 loop (R322E) had a dramatic effect over hSARM1 activity, promoting cell death levels comparable to those induced by the 'delARM' 'constitutively active' construct, which is missing the entire ARM domain (Fig. 5D). Two mutations (D314A and Q320A) that are also located at the ARM1-ARM2 loop but positioned away from the NAD+ molecule, did not induce hSARM1 activation. As expected, the control mutations E94R and K363A did not affect hSARM1 activity either. Surprisingly, mutating the bulky W103, which stacks with the NAD+ nicotinamide ring, into an alanine had only a small effect, leading to the notion that W103 does not have a critical role in NAD+ binding.
Interestingly, we did not find a density that can imply of the presence of an NAD+ molecule at the TIR domain active site, although this site is not occluded by the ARM domain (Fig. S4B).
Possibly, the BB loop, that is interacting with a neighboring ARM domain (Fig. S4B), assumes a conformation that prevents NAD+ entry into the binding cleft.
Another conspicuous distinction between the two maps, is the difference in domain-based heterogeneity in map quality in the GraFix-ed structure with respect to the relative homogeneity in the NAD+ supplemented structure (Fig. S3). While the SAM and (to a lesser extent) ARM regions are well-resolved in the GraFix-ed density map, the TIR domain is not very clear. A likely reason for the difference in map homogeneity is that while the compact arrangement in the GraFix-ed structure is artificially imposed by high glycerol concentration, the NAD+ supplement seems to induce a more natural compact folding.

Conclusions and future perspectives
Our octamer ring structure of a near-intact hSARM1 reveals an inhibited conformation, in which the catalytic TIR domains are kept apart from each other, unable to form close homodimers, which are required for their NADase activity. This inhibited conformation readily disassembles and gains most of its potential activity (Km = 27µM) during protein purification, substantiating the hypothesis that a low-affinity cellular factor inhibits hSARM1, but is lost in purification. We have tested a few candidate molecules and found that ATP inhibits the NADase activity of SARM1; however, this occurs not by promoting the compact inhibited conformation, but rather, possibly by competing over binding to the active site.
We found that NAD+ induces a dramatic conformational shift in purified hSARM1, from 'open' to 'compact' conformations, through binding to a distal allosteric site from the TIR catalytic domain.
Mutations in this allosteric site promoted hSARM1 activity in cultured cells, thereby demonstrating the key role of this site in inhibiting the NADase activity. We also found that hSARM1 is inhibited for NADase activity in vitro by NAD+, demonstrating a 'substrate inhibition mechanism'. Following these results, we propose a model for hSARM1 inhibition in homeostasis and activation under stress (Fig. 6). In this model, we suggest that hSARM1 is kept inhibited by NAD+ through its allosteric site, located at the ARM domain 'horns' junction, that induces the compact inhibited conformation. This state persists as long as NAD+ remains at normal cellular levels [these are controversial, and range between 0.1mM (39) to 0.4-0.8mM (40)(41)(42)]. However, upon a drop in the cellular NAD+ concentration below a critical threshold, such as in response to stress, NAD+ dissociates from the allosteric inhibitory site. This triggers the disassembly of the compact conformation, and the dimerization of TIR domains, which enables TIR's NADase activity and NAD+ hydrolysis. The consequence is a rapid decrease in NAD+ cellular levels, leading to energetic catastrophe and cell death.     Fig. 3F.
B) The structural effects of NAD+ and ATP were observed by cryo-EM based on appearance in 2D classification. Presented are the 10 most populated classes (those with the largest number of particles -numbers in green, 1st class is on the left) out of 50-100 from each data collection after the first round of particle picking and classification. By this analysis, the percentage of particles that present full, two-ring structure is 13% (no NAD+); 74% (5mM NAD+); and 4% (10mM ATP).   In homeostasis, the cellular NAD+ concentration is high enough and binds to an allosteric site that drives hSARM1 compact conformation. In this conformation, the catalytic TIR domains (red) are docked on ARM domains (yellow) apart from each other, unable to form close dimers required for NAD+ catalysis. When cellular NAD+ levels drop as a result of reduced NAD+ synthesis (e.g. inhibition of NMNAT1/2) or increased NAD+ consumption, the inhibiting NAD+ molecules fall off hSARM1, leading to the disintegration of the ARM-TIR outer ring assembly. Still
Assembly PCR mutagenesis (based on https://openwetware.org/wiki/Assembly_pcr) was used to introduce all the point mutations.

Protein expression and purification
For protein purification, SARM1w.t. and SARM1E642Q were expressed in HEK293F suspension cell culture, grown in FreeStyleTM 293 medium (GIBCO), at 37°C and in 8% CO2. Transfection was carried out using preheated (70°C) 40 kDa polyethyleneimine (PEI-MAX) (Polysciences) at 1mg of plasmid DNA per 1 liter of culture once cell density has reached 1*106 cells/ml. Cells were harvested 4-(in the case of SARM1w.t.) and 5-(in the case of SARM1 E642Q) days after transfection by centrifugation (10 min, 1500 x g, 4°C), re-suspended with buffer A (50mM Phosphate buffer pH 8, 400mM NaCl ,5% glycerol, 1mM DTT, 0.5mM EDTA, protease inhibitor cocktail from Roche) and lysed using a microfluidizer followed by two cycles of centrifugation (12000 x g 20 min). Supernatant was then filtered with 45µm filter and loaded onto a pre-equilibrated Ni-chelate column. The column was washed with buffer A supplemented with 25mM Imidazole until a stable baseline was achieved. Elution was then carried out in one step of 175mM Imidazole, after which protein-containing fractions were pooled and loaded onto pre-equilibrated Superdex 200 HiLoad 16/60 (GE Healthcare) for size exclusion chromatography and elution was performed with 25mM Phosphate buffer pH 8.5, 120 mM NaCl, 2.5% glycerol, and 1mM DTT. Protein-containing fractions were pooled and concentrated using a spin concentrator to 1.5 mg/ml. The concentrated proteins were either split into aliquots, flash-frozen in liquid N2 and stored at -80°C for later cryo-EM visualization and enzymatic assays, or immediately subjected to a 'GraFix' (33) procedure as follows. Ultracentrifugation was carried out using a SW41Ti rotor at 35,000rpm for 16 h at 4 °C in a 10-30% glycerol gradient (prepared with gradient master-ip® BioComp Instruments, Fredericton, Canada), with a parallel 0-0.2% glutaraldehyde gradient, and with buffer composition of 25mM Phosphate buffer pH 8.5, 120mM NaCl, and 1mM DTT. The protein solution volume that was applied to GraFix was 0.4ml. After ultracentrifugation, the 12ml gradient tube content was carefully fractionated into 0.75ml fractions, supplemented with 10mM aspartate pH 8 to quench crosslinking, using a regular pipette. Most of the cross-linked protein was found at the fractions around 18% glycerol with minor amount at the bottom of the tube (See Fig. 1B).
Analysis was made by SDS-PAGE and the dominant 2-3 fractions were pooled and diluted by 25mM Phosphate buffer pH 8.5, 120mM NaCl, and 1mM DTT, so to reach a final glycerol concentration of 2.5%. The diluted sample was then concentrated using a 100KDa cutoff Centricon® spin concentrator to reach 1mg/ml protein concentration.

Cryo-EM grids preparation
Cryo-EM grids were prepared by applying 3 μl protein samples to glow-discharged (PELCO easiGlow™ Ted Pella Inc., at 15 mA for 1 minute) holey carbon grids (Quantifoil R 1.2/1.3, Micro Tools GmbH, Germany). The grids were blotted for 4 seconds and vitrified by rapidly plunging into liquid ethane at −182 °C using Leica EM GP plunger (Leica Microsystems, Vienna, Austria).
The frozen grids were stored in liquid nitrogen until the day of cryo-EM data collection.

Cryo-EM data acquisition and processing
In this paper we present data that was collected with three separate cryo-electron microscopes: 1) F30 Polara in Ben-Gurion University, Israel was used for all sample preparation optimization. It was also used to collect data sets without and with potential inhibitors (shown in Fig. 1C and the ATP and NMN supplemented classes in Fig. 4B). Finally, it was used for data collection and the 3D reconstructions presented in Fig. 1D.
Samples were imaged under low-dose conditions on a FEI Tecnai F30 Polara microscope (FEI, Eindhoven) operating at 300kV. Datasets were collected using SerialEM (44) on a K2 Summit direct electron detector fitted behind an energy filter (Gatan Quantum GIF) with a calibrated pixel size of 1.1Å. The energy filter was set to remove electrons > ±10eV from the zero-loss peak energy. The defocus range was set from -1.0μm to -2.5μm. The K2 summit camera was operated in counting mode at a dose rate of 8 electrons/pixel/second on the camera. Each movie was dose fractionated into 50 image frames, with total electron dose of 80ē/Å2. Dose-fractionated image stacks were aligned using MotionCorr2 (45), and their defocus values estimated by Gctf (46). The sum of the aligned frames was used for further processing and the rest of the processing was done in Cryosparc V2 (47). Particles were auto-picked and subjected to local motion correction to correct for beam-induced drift and then 2D classification with 50 classes. The best (based on shape, number of particles and resolution) classes were manually selected containing 5459 (for hSARM1E642Q) and 43868 (for SAM1-2) particles. 1 initial 3D reference was prepared from all particles and 3D refinement imposing C8 symmetry resulted the final map. Further data processing was conducted using the cryoSPARC suite. Movies were motioncorrected and contrast transfer functions were fitted. Templates for auto-picking were generated by 2D classification of auto picked particles. For the GraFix-ed data, template-based auto-picking produced a total of 658,575 particles, from which 147,232 were selected based on iterative reference-free 2D classifications for reconstruction of the GraFix-ed structure. In the case of the NAD+ supplemented data, a total of 335,526 particles were initially picked, from which 159,340 were selected based on iterative reference-free 2D classifications for reconstruction. Initial maps of both GraFix-ed and NAD+ supplemented hSARM1 were calculated using Ab-initio reconstruction and high-resolution maps were obtained by imposing C8-symmetry in non-uniform 3D refinement. Working maps were locally filtered based on local resolution estimates.
3) Talos Glacios in EMBL, Grenoble, France was used for data collection and the comparison of NAD+ supplemented and not-supplemented samples (Fig. 4A,B).
Frozen grids were loaded into a 200kV Talos Glacios (ThermoFisher) electron microscope equipped with a Falcon3 direct electron-counting camera (ThermoFisher). Cryo-EM data were acquired with EPU software (FEI) at a nominal magnification of ×120,000, with a pixel size of

Calculation of SARM1 kinetic parameters
For Vmax and Km determination, the NADase activity assay was performed with several different NAD+ substrate concentrations and sampled in constant time points. For each NAD+ concentration, linear increase zone was taken for slope (V0) calculation. All data were than fitted to the Michaelis-Menten equation using non-linear curve fit in GraphPad Prism software. Kcat was calculated by dividing the Vmax with protein molar concentration.

GraFix-ed map
The monomers of known octameric X-ray structures of the hSARM1 SAM1-2 domains (PDBs 6qwv and 6o0s) were superimposed by the CCP4 (53) program GESAMT (54) (57). The quality of the high resolution GraFix-ed density map was sufficient for assignment of side chains for all ARM domain residues. The TIR domain BB loop region (a.a 595-607) was built to fit a relatively poor density map in a conformation different to those observed in X-ray structures. The model was refined using both REFMAC5 (58). Side chains of some Lys residues had blobs of undescribed density attached to them. These were modelled as glutaraldehyde ligands. Geometrical restraints for the di-glutaraldehyde molecule and its links to side chains of Lys residues were prepared using JLIGAND (59). The dictionary file was manually edited to allow links to more than a single lysine residue.

NAD-supplemented map
Originally, the refined full-length GraFix-ed model was positioned by MR into the NADsupplemented density map, but differences in the relative positions of the hSARM1 domains were apparent. Therefore, MR search was conducted for separate hSARM1 domains. The SAM Model was positioned with a score of 0.653 into this map. With the fixed SAM Model the TIR Model 1 was found with score of 0.594. With fixed SAM and TIR Models, the ARM domain from the GraFix model was found with score of 0.623. The BB-loop of the TIR domain was built into welldefined electron density map in conformation not observed in any of the X-ray structures and different to that in the GraFix-ed model. A low sigma cutoff map allowed modelling of the loop connecting the SAM and TIR domains. Inspection of the maps indicated NAD+ binding accompanied by structural re-arrangement of the ARM1-ARM2 linker region (a.a. 312-324).

Accession numbers
Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 6ZFX, 6ZZ7, 6ZG0, 6ZG1, and in the EMDB with accession numbers 11187, 11586, 11190, 11191 for the GraFix-ed, NAD+ supplemented, not treated, and SAM1-2 models and maps, respectively.