Structure and dynamics of the Arabidopsis O-fucosyltransferase SPINDLY

Kumar and colleagues report cryo-EM structures of ArabidopsisO-fucosyltransferase SPINDLY in its apo and GDP-fucose bound states, revealing distinct active-site features enabling GDP-fucose recognition and surprisingly dynamic conformations that regulate its enzymatic activity. 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.

Automated summary

Kumar and colleagues have revealed the distinct features of Arabidopsis O-fucosyltransferase SPINDLY (SPY) through cryo-EM structures. They found that SPY has unique active-site features and dynamic conformations that regulate its enzymatic activity. The study also showed that the N-terminal peptide and TPRs in SPY play important roles in inhibiting and regulating its activity.