National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
United States
Citation
Journal: Nat Commun / Year: 2023 Title: Structure and dynamics of the Arabidopsis O-fucosyltransferase SPINDLY. Authors: Shivesh Kumar / Yan Wang / Ye Zhou / Lucas Dillard / Fay-Wei Li / Carly A Sciandra / Ning Sui / Rodolfo Zentella / Emily Zahn / Jeffrey Shabanowitz / Donald F Hunt / Mario J Borgnia / ...Authors: Shivesh Kumar / Yan Wang / Ye Zhou / Lucas Dillard / Fay-Wei Li / Carly A Sciandra / Ning Sui / Rodolfo Zentella / Emily Zahn / Jeffrey Shabanowitz / Donald F Hunt / Mario J Borgnia / Alberto Bartesaghi / Tai-Ping Sun / Pei Zhou / Abstract: SPINDLY (SPY) in Arabidopsis thaliana is a novel nucleocytoplasmic protein O-fucosyltransferase (POFUT), which regulates diverse developmental processes. Sequence analysis indicates that SPY is ...SPINDLY (SPY) in Arabidopsis thaliana is a novel nucleocytoplasmic protein O-fucosyltransferase (POFUT), which regulates diverse developmental processes. Sequence analysis indicates that SPY is distinct from ER-localized POFUTs and contains N-terminal tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain resembling the O-linked-N-acetylglucosamine (GlcNAc) transferases (OGTs). However, the structural feature that determines the distinct enzymatic selectivity of SPY remains unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of SPY and its complex with GDP-fucose, revealing distinct active-site features enabling GDP-fucose instead of UDP-GlcNAc binding. SPY forms an antiparallel dimer instead of the X-shaped dimer in human OGT, and its catalytic domain interconverts among multiple conformations. Analysis of mass spectrometry, co-IP, fucosylation activity, and cryo-EM data further demonstrates that the N-terminal disordered peptide in SPY contains trans auto-fucosylation sites and inhibits the POFUT activity, whereas TPRs 1-5 dynamically regulate SPY activity by interfering with protein substrate binding.
Model: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 40 / Pretreatment - Type: PLASMA CLEANING
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 289.15 K / Instrument: LEICA EM GP
-
Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Average electron dose: 52.4 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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