Structural insights into mechanism and specificity of the plant protein O-fucosyltransferase SPINDLY

Abstract

Arabidopsis glycosyltransferase family 41 (GT41) protein SPINDLY (SPY) plays pleiotropic roles in plant development. Despite the amino acid sequence is similar to human O-GlcNAc transferase, Arabidopsis SPY has been identified as a novel nucleocytoplasmic protein O-fucosyltransferase. SPY-like proteins extensively exist in diverse organisms, indicating that O-fucosylation by SPY is a common way to regulate intracellular protein functions. However, the details of how SPY recognizes and glycosylates substrates are unknown. Here, we present a crystal structure of Arabidopsis SPY/GDP complex at 2.85 Å resolution. SPY adopts a head-to-tail dimer. Strikingly, the conformation of a ‘catalytic SPY’/GDP/‘substrate SPY’ complex formed by two symmetry-related SPY dimers is captured in the crystal lattice. The structure together with mutagenesis and enzymatic data demonstrate SPY can fucosylate itself and SPY’s self-fucosylation region negatively regulates its enzyme activity, reveal SPY’s substrate recognition and enzyme mechanism, and provide insights into the glycan donor substrate selection in GT41 proteins.

Structure and dynamics of the Arabidopsis O-fucosyltransferase SPINDLY

“…Superimposition of the catalytic domain of the crystal structure of the SPY/GDP complex with that of the cryo-EM structure of the SPY/ GDP-fucose complex reveals a consistent binding pose of the GDP moiety between the crystal and cryo-EM structures and that the receptor serine sidechain hydroxyl group from the auto-inhibitory Nterminal SPY peptide is located in proximity to the C1 carbon of the GDP-fucose ring (Supplementary Fig. 13), lending further support to the proposed mechanism of a substrate-assisted SN2 reaction proposed here and by Zhu et al 39 . As recent proteomic analysis of SPY substrates did not reveal a distinct pattern around the O-fucosylation sites, except the enrichment of serine residues 37,38 , how SPY selectively recognizes diverse peptide sequences requires further investigation.…”

“…The superimposition of the crystal structure with the cryo-EM structure shows an overall excellent agreement of the structural features. Interestingly, although Zhu et al concluded that it is unlikely for SPY TPRs to display any conformational flexibility 39 , our cryo-EM analysis readily reveals the presence of multiple conformations of the TPRs and catalytic domain orientation (Fig. 3 ).…”

“…While this manuscript was under revision, a crystal structure of auto-inhibited Arabidopsis SPY in complex with GDP was reported 39 . The superimposition of the crystal structure with the cryo-EM structure shows an overall excellent agreement of the structural features.…”

Structure-based virtual screening identifies small-molecule inhibitors of O-fucosyltransferase SPINDLY in Arabidopsis

“…We analyzed how SOFTI interacts with specific residues within the solved structure of the substrate-binding pocket of SPY. For our screen, we used a structure predicted by AlphaFold; however, the structure of SPY has recently been determined by crystallography and cryo-EM ( Zhu et al 2022 ; Kumar et al 2023 ). We performed molecular docking between SOFTI and the crystal structure of SPY.…”

“…1 ). Further, the molecular docking of SOFTI to the catalytic pocket of SPY showed that SOFTI interacts with multiple key amino acid residues that interact with the donor substrate GDP-fucose according to the crystal and cryo-EM structures ( Zhu et al 2022 ; Kumar et al 2023 ; Fig. 4, A to D ).…”