PXD033528-1

PXD033528 is an original dataset announced via ProteomeXchange.

Title	A Conformation specific Nanobody Targeting the NMN-activated State of SARM1
Description	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 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.
HostingRepository	PRIDE
AnnounceDate	2022-11-29
AnnouncementXML	Submission_2022-11-29_05:18:55.092.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	GoranStjepanovic
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	iodoacetamide derivatized residue
Instrument	Orbitrap Eclipse

Revision	Datetime	Status	ChangeLog Entry
0	2022-04-28 01:36:23	ID requested
⏵ 1	2022-11-29 05:18:55	announced

Dataset with its publication pending

submitter keyword: SARM1, ADP ribosyl cyclase, XL-MS, HDX-MS

GoranStjepanovic
contact affiliation	Kobilka Institute of Innovative Drug Discovery, School ofMedicine, The Chinese University of Hong Kong, Shenzhen, China, 518172
contact email	goranstjepanovic@cuhk.edu.cn
lab head
GoranStjepanovic
contact affiliation	CUHKSZ
contact email	goranstjepanovic@cuhk.edu.cn
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD033528
     1. Label: PRIDE project
     2. Name: A Conformation specific Nanobody Targeting the NMN-activated State of SARM1