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Research Project: Strategies to Support Resilient Agricultural Systems of the Southeastern U.S.

Location: Plant Science Research

Title: Structure and dynamics of the Arabidopsis O-fucosyltransferase SPINDLY

Author
item KUMAR, SHIVESH - Duke University
item WANG, YAN - Duke University
item ZHOU, YE - Duke University
item DILLARD, LUCAS - National Institute Of Environmental Health Sciences (NIEHS, NIH)
item LI, FAY-WEI - Cornell University
item SCIANDRA, CARLY - Duke University
item SUI, NING - Duke University
item ZENTELLA GOMEZ, RODOLFO - Duke University
item BORGNIA, MARIO - National Institute Of Environmental Health Sciences (NIEHS, NIH)
item BARTESAGHI, ALBERTO - Duke University
item SUN, TAI-PING - Duke University
item ZHOU, PEI - Duke University

Submitted to: Nature Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/4/2023
Publication Date: 3/20/2023
Citation: Kumar, S., Wang, Y., Zhou, Y., Dillard, L., Li, F., Sciandra, C., Sui, N., Zentella Gomez, R., Borgnia, M., Alberto Bartesaghi, A., Sun, T., Zhou, P. 2023. Structure and dynamics of the Arabidopsis O-fucosyltransferase SPINDLY. Nature Communications. 14:1538. https://doi.org/10.1038/s41467-023-37279-1.
DOI: https://doi.org/10.1038/s41467-023-37279-1

Interpretive Summary: SPINDLY (SPY) in Arabidopsis thaliana is a novel nuclear and cytoplasmic enzyme that attaches fucose (Fuc), a sugar molecule, to protein targets. By this action, SPY regulates diverse developmental processes. Sequence analysis indicates that AtSPY is distinct from enzymes with the same enzymatic activity found in other cellular compartments. AtSPY, has a striking resemblance to a related enzyme that instead of Fuc, it attaches a different sugar, N-acetylglucosamine (GlcNAc), to proteins. The sequences of these proteins are so similar that they were believed to have the same enzymatic activity. Here, we report the 3D structure of AtSPY in a complex with its donor substrate GDP-Fuc, obtained by cryo-electron microscopy. Multiple states of the protein were visualized, which allowed us to assign structural and enzymatic functions to different parts of AtSPY. Our results also allowed us to pinpoint the structural differences in AtSPY that confers specificity for the transfer of Fuc, and not GlcNAc, to proteins.

Technical Abstract: SPINDLY (SPY) in Arabidopsis thaliana is a novel nucleocytoplasmic protein Ofucosyltransferase (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.