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PXD033528-1

PXD033528 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleA Conformation specific Nanobody Targeting the NMN-activated State of SARM1
DescriptionUpon 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 30 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.
HostingRepositoryPRIDE
AnnounceDate2022-11-29
AnnouncementXMLSubmission_2022-11-29_05:18:55.092.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterGoranStjepanovic
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListiodoacetamide derivatized residue
InstrumentOrbitrap Eclipse
Dataset History
RevisionDatetimeStatusChangeLog Entry
02022-04-28 01:36:23ID requested
12022-11-29 05:18:55announced
Publication List
Dataset with its publication pending
Keyword List
submitter keyword: SARM1, ADP ribosyl cyclase, XL-MS, HDX-MS
Contact List
GoranStjepanovic
contact affiliationKobilka Institute of Innovative Drug Discovery, School ofMedicine, The Chinese University of Hong Kong, Shenzhen, China, 518172
contact emailgoranstjepanovic@cuhk.edu.cn
lab head
GoranStjepanovic
contact affiliationCUHKSZ
contact emailgoranstjepanovic@cuhk.edu.cn
dataset submitter
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