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Structure and dynamics of the Arabidopsis O-fucosyltransferase SPINDLY

ABSTRACT: SPINDLY (SPY) in Arabidopsis thaliana is a novel nucleocytoplasmic protein O-fucosyltransferase (POFUT), which regulates diverse developmental processes. Sequence analysis indicates that AtSPY 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 AtSPY and its complex with GDP-fucose, revealing distinct active-site features enabling GDP-fucose instead of UDP-GlcNAc binding. AtSPY forms an antiparallel dimer instead of the X-shaped dimer in human OGT, and its catalytic domain dynamically interconverts among inward, middle, and outward states. The entire eleven TPRs are visible in the middle state, with TPR1 of one subunit touching down on the catalytic domain of the other subunit of the AtSPY dimer, whereas the densities of TPRs 1-5 are absent or severely attenuated in the inward and outward states. Analysis of mass spectrometry, co-IP, in planta fucosylation activity, and cryo-EM data further demonstrates that the N-terminal disordered peptide in SPY contains trans auto-fucosylation sites and inhibits its catalytic activity, whereas TPRs 1-5 regulate SPY activity by interfering with protein substrate binding.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Nicotiana Tabacum (common Tobacco)

SUBMITTER: Jeffrey Shabanowitz

LAB HEAD: Jeffrey Shabanowitz

PROVIDER: PXD040480 | Pride | 2023-02-28

REPOSITORIES: Pride

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