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

Abstract

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 descriptionIt 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.

Neurotoxins subvert the allosteric activation mechanism of SARM1 to induce neuronal loss

“…NAD + is generated from Nam in two steps: the rate-limiting conversion of Nam to NMN by NAMPT, followed by synthesis of NAD + from NMN by NMNAT2 and its paralogs (Sauve, 2008). NMN and NAD + compete to bind an allosteric pocket that modulates SARM1 autoinhibition, rendering SARM1 a metabolic sensor that responds to an elevated NMN/NAD + ratio (Figley et al, 2021;Jiang et al, 2020;Sporny et al, 2020). As such, experimental manipulations that alter the apparent concentration of either metabolite, whether by short-circuiting the usual synthesis pathway or by interjecting an NMN mimetic, directly affect SARM1 activity and its consequences (Di Stefano et al, 2017;Sasaki et al, 2009;Zhao et al, 2019).…”

“…In healthy neurons, SARM1 is maintained in an autoinhibited state via multiple intra-and intermolecular interactions (Shen et al, 2021), including binding of the N-terminal ARM domain to the C-terminal TIR-domain (Summers et al, 2016). SARM1 autoinhibition is regulated by an allosteric binding site within the autoinhibitory ARM domain that can bind either nicotinamide adenine dinucleotide (NAD + ) (Jiang et al, 2020;Sporny et al, 2020) or its precursor, nicotinamide mononucleotide (NMN) (Figley et al, 2021). Axon injury leads to loss of the NAD + biosynthetic enzyme NMNAT2 (Gilley et al, 2010), resulting in an increased NMN/NAD + ratio that favors NMN binding to the allosteric site (Figley et al, 2021).…”

Early loss of endogenous NAD+ following rotenone treatment leads to Sarm1 activation that is ameliorated by PARP inhibition

“…We report here that rotenone induced temporal regulation of NAD + levels lead to a sequential triggering of events that ultimately cause cell death. Our study indicates that rotenone treatment results in an early loss of NAD + that is accompanied by mitochondrial dysfunction and PARP1 hyperactivation that may serve as the “biological trigger” of Sarm1 induction which has been previously shown to be allosterically inhibited by NAD + using in vitro systems [29,30,31]. In concurrence with these observations, replenishing NAD + levels by pre-incubation of cells with the PARP inhibitor PJ34 or Olaparib restored cellular NAD + levels, prevented Sarm1 activation and significantly reduced cell death following rotenone treatment.…”

SARM1-Dependent Axon Degeneration: Nucleotide Signaling, Neurodegenerative Disorders, Toxicity, and Therapeutic Opportunities

“…The ARM:TIR interactions regulate SARM1 by preventing TIR self-association, and disruption of the ARM:TIR interface by site-directed mutagenesis produces a constitutively active SARM1 (Bratkowski and others 2020). In cryo-EM structures of human SARM1 and crystal structures of Drosophila SARM1, the ARM domain revealed an unusual compact superhelix with the N-and C-terminal regions collapsed around the allosteric activator NMN, or the allosteric inhibitors NAD + and NaMN (Figure 5C) (Figley and others 2021;Jiang and others 2020;Sasaki and others 2021;Sporny and others 2020). In two recent cryo-EM structures of hSARM1 incubated with NMN, the ARM domains have undergone a significant reorientation with respect to the octameric SAM domain ring (Hou and others 2022;Shi and others 2022).…”