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Multiple Domain Interfaces Mediate SARM1 Autoinhibition

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Multiple Domain Interfaces Mediate SARM1 Autoinhibition Multiple domain interfaces mediate SARM1 autoinhibition Chen Shena,b,1, Mihir Vohrac,1, Pengfei Zhanga,b, Xianrong Maoc, Matthew D. Figleyd, Jian Zhuc, Yo Sasakic, Hao Wua,b,2, Aaron DiAntoniod,e,2, and Jeffrey Milbrandtc,e,2 aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; bProgram in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115; cDepartment of Genetics, Washington University School of Medicine, St. Louis, MO 63110; dDepartment of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110; and eNeedleman Center for Neurometabolism and Axonal Therapeutics, Washington University School of Medicine, St. Louis, MO 63110 Contributed by Hao Wu, December 17, 2020 (sent for review November 10, 2020; reviewed by Robert W. Burgess and Pingwei Li) Axon degeneration is an active program of self-destruction medi- α-motif (SAM) domains, and a Toll/interleukin-1 receptor (TIR) ated by the protein SARM1. In healthy neurons, SARM1 is auto- domain (5). While TIR domains are canonically known for scaf- inhibited and, upon injury autoinhibition is relieved, activating the folding functions (19), the SARM1 TIR domain is the founding + SARM1 enzyme to deplete NAD and induce axon degeneration. member of an evolutionarily conserved family of NAD+ hydro- SARM1 forms a homomultimeric octamer with each monomer lases (NADases) (18, 20–22). SARM1 enzyme activity is necessary composed of an N-terminal autoinhibitory ARM domain, tandem for axon degeneration (20), and active SARM1 TIR is sufficient to SAM domains that mediate multimerization, and a C-terminal TIR induce degeneration in otherwise healthy neurons (23). To control domain encoding the NADase enzyme. Here we discovered multi- ple intramolecular and intermolecular domain interfaces required this prodegenerative activity, SARM1 regulation must incorporate for SARM1 autoinhibition using peptide mapping and cryo-electron two key features: 1) enzymatic activity must be inhibited in healthy microscopy (cryo-EM). We identified a candidate autoinhibitory neurons, and 2) injury- or disease-induced prodegenerative signals region by screening a panel of peptides derived from the SARM1 must relieve this autoinhibition. Autoinhibition is a common ARM domain, identifying a peptide mediating high-affinity inhibi- mechanism for tightly controlling protein activity, effectively tion of the SARM1 NADase. Mutation of residues in full-length maintaining an “off” state at equilibrium and rapidly responding SARM1 within the region encompassed by the peptide led to loss to stimuli to induce the “on” state (24–26). The N-terminal ARM NEUROSCIENCE of autoinhibition, rendering SARM1 constitutively active and in- domain is required for autoinhibition of SARM1, as deletion + ducing spontaneous NAD and axon loss. The cryo-EM structure of mutants lacking the entire N terminus are constitutively active, SARM1 revealed 1) a compact autoinhibited SARM1 octamer in leading to spontaneous axon degeneration (5). Moreover, the which the TIR domains are isolated and prevented from oligomer- N-terminal ARM region physically associates with the enzymatic ization and enzymatic activation and 2) multiple candidate auto- TIR domain of SARM1, suggesting that the N terminus may inhibitory interfaces among the domains. Mutational analysis mediate autoinhibition via direct inhibition of the TIR NADase demonstrated that five distinct interfaces are required for auto- inhibition, including intramolecular and intermolecular ARM-SAM interfaces, an intermolecular ARM-ARM interface, and two ARM- Significance TIR interfaces formed between a single TIR and two distinct ARM domains. These autoinhibitory regions are not redundant, as point Axon degeneration is an active program of subcellular self- mutants in each led to constitutively active SARM1. These studies destruction that drives pathology in the injured and diseased define the structural basis for SARM1 autoinhibition and may en- nervous system. SARM1 is an inducible NAD+ hydrolase and the able the development of SARM1 inhibitors that stabilize the central executioner of axon loss. In healthy axons, the SARM1 autoinhibited state. NADase is autoinhibited. With injury or disease, this auto- inhibition is relieved and SARM1 depletes NAD+, inducing a neurodegeneration | axonopathy | metabolism | neuropathy | NMN metabolic crisis and subsequent axon loss. Here we combine peptide screening, cryo-electron microscopy, and site-directed xon degeneration is a key feature of neurodegeneration in mutagenesis with analysis of axonal metabolomics and axon Awhich injured and diseased axons activate a program of degeneration to define five domain interactions within and subcellular self-destruction (1, 2). Mechanistically, the choice across SARM1 protomers that are required to maintain an in- active SARM1 octamer. These structural insights may enable between axon survival and degeneration depends on the balance the development of SARM1 inhibitors that stabilize this auto- between axon maintenance and axon destruction proteins (3). inhibited conformation and thereby block axon degeneration. SARM1 (sterile α and TIR motif-containing protein 1) is the essential prodegenerative protein in this pathway. Loss of SARM1 Author contributions: C.S., M.V., H.W., A.D., and J.M. designed research; C.S., M.V., P.Z., is profoundly axoprotective in animal models of nerve injury (4, 5), X.M., M.D.F., J.Z., and Y.S. performed research; P.Z. contributed new reagents/analytic peripheral neuropathy (6–8), traumatic brain injury (9–11), and tools; C.S., M.V., X.M., M.D.F., J.Z., Y.S., H.W., A.D., and J.M. analyzed data; and C.S., M.V., glaucoma (12). SARM1 can also drive neuronal cell death. SARM1 H.W., A.D., and J.M. wrote the paper. knockout (KO) inhibits neuronal loss induced by mitochondrial Reviewers: R.W.B., The Jackson Laboratory; and P.L., Texas A&M University. dysfunction (13) in a mouse model of TDP-43–associated amyo- Competing interest statement: A.D. and J.M. are cofounders, scientific advisory board members, and shareholders of Disarm Therapeutics, a wholly owned subsidiary of Eli Lilly trophic lateral sclerosis (ALS) (14) and in multiple models of & Co. Y.S. is a consultant to Disarm Therapeutics. photoreceptor neurodegeneration (15, 16). These studies demonstrate Published under the PNAS license. that traumatic, metabolic, toxic, genetic, and neuroinflammatory 1C.S. and M.V. contributed equally to this work. insults can all activate SARM1 and highlight the importance of 2To whom correspondence may be addressed. Email: [email protected], elucidating the mechanism of SARM1 regulation. [email protected], or [email protected]. SARM1 functions as an octamer (17, 18) in which each This article contains supporting information online at https://www.pnas.org/lookup/suppl/ monomer is composed of a mitochondrial targeting sequence, an doi:10.1073/pnas.2023151118/-/DCSupplemental. N-terminal domain with armadillo repeats (ARM), two sterile Published January 18, 2021. PNAS 2021 Vol. 118 No. 4 e2023151118 https://doi.org/10.1073/pnas.2023151118 | 1of9 Downloaded by guest on September 30, 2021 (27). However, the detailed molecular basis for this autoinhibition These results suggested that peptide 5 interacted with SARM1 in remains elusive. a specific manner that led to its functional inhibition (SI Appendix, Here we have combined functional and structural analysis Fig. S1 C and D). of SARM1 to define the domain interfaces required for auto- The SARM1 peptide 5 extends from arginine at 244 to histi- inhibition. To identify a region in the ARM domain that medi- dine at 269. The most striking feature of this sequence is a highly ates autoinhibition, we screened a collection of peptides that tile evolutionarily conserved region of hydrophobic residues. We this domain and identified a single peptide that potently inhibits used MUSCLE to conduct multiple sequence alignment for 439 SARM1 NADase enzyme activity. This peptide encompasses an metazoan sequences with high homology to human SARM1. evolutionarily conserved sequence of hydrophobic residues that This analysis shows the conserved residues at each position are required for its inhibitory activity. Accordingly, mutation of where the tall black letters from W253 to F259 indicate these these required hydrophobic residues within the full-length SARM1 highly evolutionarily conserved hydrophobic residues (Fig. 1B). protein disrupts autoinhibition, resulting in a constitutively active Reasoning that these residues may be conserved because they + SARM1 enzyme that cleaves NAD and induces axon degenera- are functionally important for inhibition, we designed a mutant tion in the absence of an injury stimulus. Expression of a protein variant of the peptide, in which the conserved hydrophobic res- composed of a concatemer of this hydrophobic region in neurons + idues were mutated to hydrophilic residues (W253T/L254S/ effectively blocks SARM1-induced NAD lossandaxondegen- F255T/L257S/F259T). We compared the mutant and wild-type eration. To understand SARM1 autoinhibition more comprehen- peptides using the in vitro NADase assay and found that peptide sively, we solved a cryo-electron microscopy (cryo-EM) structure 5 blocked SARM1:SAM-TIR activity in a dose-dependent of the SARM1 octamer and identified five intramolecular and manner with an IC50 of about 20 nM (Fig. 1C). Strikingly, the intermolecular interfaces occurring between domains of SARM1 mutant peptide
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