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A conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1.

ABSTRACT: Sterile alpha (SAM) and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1) is an autoinhibitory NAD-consuming enzyme that is activated by the accumulation of nicotinamide mononucleotide (NMN) during axonal injury. Its activation mechanism is not fully understood. Here, we generate a nanobody, Nb-C6, that specifically recognizes NMN-activated SARM1. Nb-C6 stains only the activated SARM1 in cells stimulated with CZ-48, a permeant mimetic of NMN, and partially activates SARM1 in vitro and in cells. Cryo-EM of NMN/SARM1/Nb-C6 complex shows an octameric structure with ARM domains bending significantly inward and swinging out together with TIR domains. Nb-C6 binds to SAM domain of the activated SARM1 and stabilized its ARM domain. Mass spectrometry analyses indicate that the activated SARM1 in solution is highly dynamic and that the neighboring TIRs form transient dimers via the surface close to one BB loop. We show that Nb-C6 is a valuable tool for studies of SARM1 activation.

SUBMITTER: Hou YN

PROVIDER: S-EPMC9780360 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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A conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1.

Hou Yun Nan YN Cai Yang Y Li Wan Hua WH He Wei Ming WM Zhao Zhi Ying ZY Zhu Wen Jie WJ Wang Qiang Q Mai Xinyi X Liu Jun J Lee Hon Cheung HC Stjepanovic Goran G Zhang Hongmin H Zhao Yong Juan YJ

Nature communications 20221222 1

Sterile alpha (SAM) and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1) is an autoinhibitory NAD-consuming enzyme that is activated by the accumulation of nicotinamide mononucleotide (NMN) during axonal injury. Its activation mechanism is not fully understood. Here, we generate a nanobody, Nb-C6, that specifically recognizes NMN-activated SARM1. Nb-C6 stains only the activated SARM1 in cells stimulated with CZ-48, a permeant mimetic of NMN, and partially activates SARM1 in vitro and ...[more]

PMID: 36550129

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Project description:SARM1 is an inducible NAD+ hydrolase that is the central executioner of pathological axon loss. Recently, we elucidated the molecular mechanism of SARM1 activation, demonstrating that SARM1 is a metabolic sensor regulated by the levels of NAD+ and its precursor, nicotinamide mononucleotide (NMN), via their competitive binding to an allosteric site within the SARM1 N-terminal ARM domain. In healthy neurons with abundant NAD+, binding of NAD+ blocks access of NMN to this allosteric site. However, with injury or disease the levels of the NAD+ biosynthetic enzyme NMNAT2 drop, increasing the NMN/ NAD+ ratio and thereby promoting NMN binding to the SARM1 allosteric site, which in turn induces a conformational change activating the SARM1 NAD+ hydrolase. Hence, NAD+ metabolites both regulate the activation of SARM1 and, in turn, are regulated by the SARM1 NAD+ hydrolase. This dual upstream and downstream role for NAD+ metabolites in SARM1 function has hindered mechanistic understanding of axoprotective mechanisms that manipulate the NAD+ metabolome. Here we reevaluate two methods that potently block axon degeneration via modulation of NAD+ related metabolites, 1) the administration of the NMN biosynthesis inhibitor FK866 in conjunction with the NAD+ precursor nicotinic acid riboside (NaR) and 2) the neuronal expression of the bacterial enzyme NMN deamidase. We find that these approaches not only lead to a decrease in the levels of the SARM1 activator NMN, but also an increase in the levels of the NAD+ precursor nicotinic acid mononucleotide (NaMN). We show that NaMN inhibits SARM1 activation, and demonstrate that this NaMN-mediated inhibition is important for the long-term axon protection induced by these treatments. Analysis of the NaMN-ARM domain co-crystal structure shows that NaMN competes with NMN for binding to the SARM1 allosteric site and promotes the open, autoinhibited configuration of SARM1 ARM domain. Together, these results demonstrate that the SARM1 allosteric pocket can bind a diverse set of metabolites including NMN, NAD+, and NaMN to monitor cellular NAD+ homeostasis and regulate SARM1 NAD+ hydrolase activity. The relative promiscuity of the allosteric site may enable the development of potent pharmacological inhibitors of SARM1 activation for the treatment of neurodegenerative disorders.

Dataset Information

A conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1.

Publications

A conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1.

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