A Conformation-specific Nanobody Targeting the NMN-activated State of SARM1

Hou, Y. N.; Cai, Y.; Li, W. H.; He, W. M.; Zhao, Z. Y.; Zhu, W. J.; Wang, Q.; Liu, J.; Lee, H. C.; Goran, S.; Zhang, H.; Zhao, Y. J.

doi:10.1101/2022.03.25.485784

bioRxiv · 10.1101/2022.03.25.485784

A Conformation-specific Nanobody Targeting the NMN-activated State of SARM1

Hou, Y. N.·Cai, Y.·Li, W. H.·He, W. M.·Zhao, Z. Y.·Zhu, W. J.·Wang, Q.·Liu, J.·Lee, H. C.·Goran, S.·Zhang, H.·Zhao, Y. J.

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Abstract

Upon axonal injury, Sterile alpha (SAM) and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1) is activated by nicotinamide mononucleotide (NMN) to deplete NAD and consequently promote the process of axon degeneration (AxD). Currently, only the inactive form of SARM1 in its auto-inhibitory conformation has been resolved. The flexibility of the enzymatically active form of SARM1 has so far precluded its structural determination. To solve the problem, we generated a stabilizing nanobody, Nb-C6, that specifically recognized only the NMN-activated form of SARM1. The conformation specificity was verified by immunoprecipitation and surface plasmon resonance. Fluorescently labeled Nb-C6 could immunostain only the activated SARM1 in cells stimulated with CZ-48, a permeant mimetic of NMN. Expression of Nb-C6 in live cells resulted in stabilization of the active form of the endogenous and exogenous SARM1, producing and elevating cellular levels of cyclic ADP-ribose, a calcium messenger. Cryo-EM of the NMN-activated SARM1 complexed by Nb-C6 showed an octameric structure resembling a "blooming lotus" with the ARM domains bending significantly inward and swinging out together with the TIR domains to form the "petals of the lotus". Nb-C6 bound to the SAM domain of the activated SARM1 and stabilized its Armadillo repeat motif domain. Analyses using hydrogen-deuterium exchange mass spectrometry (HDX-MS), and cross-linking MS (XL-MS) indicate that the activated SARM1 is highly dynamic and flexible and the neighboring TIRs form dimers via the surface close to one BB loop. The Nanobody is thus a valuable tool for delineating the mechanism of activation of SARM1 in AxD and other cellular processes.

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Hou, Y. N., Cai, Y., Li, W. H., He, W. M., Zhao, Z. Y., Zhu, W. J., Wang, Q., Liu, J., Lee, H. C., Goran, S., Zhang, H., Zhao, Y. J.. 2022-03-26. A Conformation-specific Nanobody Targeting the NMN-activated State of SARM1. https://doi.org/10.1101/2022.03.25.485784

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