Cryo-EM structure provides insights into the dimer arrangement of the O-linked β-N-acetylglucosamine transferase OGT

Meek, Richard W.; Blaza, James N.; Busmann, Jil A.; Alteen, Matthew G.; Vocadlo, David J.; Davies, G.J.

doi:10.1038/s41467-021-26796-6

Cited by 28 publications

(33 citation statements)

References 37 publications

(74 reference statements)

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“…The overall structure of Arabidopsis SPY monomer shows little similarity with that of human ER-localized protein O-fucosyltransferase POFUT1/2 33,34 but most resembles that of human OGT 35,36 (Supplementary Fig. 5).…”

Section: The Structure Of Spy/gdp Complexmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: The Structure Of Spy/gdp Complexmentioning

confidence: 99%

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

et al. 2022

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“…For example, the generation of a monomeric form of human lysyl hydrolase 3 (LH3), which is a multifunctional GTase, results in deterioration of enzymatic activity [ 60 ]. Destabilization of the homodimeric interface area by disease causing mutations in O-linked beta-N-acetylglucosamine transferase (OGT) was found to decrease its catalytic activity [ 61 ]. Additionally, disruption of homodimerization for the dual glycosyltransferase 1 (dGT1) also lead to a significantly decrease in its GlcNAc-T activity [ 36 ].…”

Section: Discussionmentioning

confidence: 99%

Structural basis for matriglycan synthesis by the LARGE1 dual glycosyltransferase

Katz

Diskin

2022

PLoS ONE

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LARGE1 is a bifunctional glycosyltransferase responsible for generating a long linear polysaccharide termed matriglycan that links the cytoskeleton and the extracellular matrix and is required for proper muscle function. This matriglycan polymer is made with an alternating pattern of xylose and glucuronic acid monomers. Mutations in the LARGE1 gene have been shown to cause life-threatening dystroglycanopathies through the inhibition of matriglycan synthesis. Despite its major role in muscle maintenance, the structure of the LARGE1 enzyme and how it assembles in the Golgi are unknown. Here we present the structure of LARGE1, obtained by a combination of X-ray crystallography and single-particle cryo-EM. We found that LARGE1 homo-dimerizes in a configuration that is dictated by its coiled-coil stem domain. The structure shows that this enzyme has two canonical GT-A folds within each of its catalytic domains. In the context of its dimeric structure, the two types of catalytic domains are brought into close proximity from opposing monomers to allow efficient shuttling of the substrates between the two domains. Together, with putative retention of matriglycan by electrostatic interactions, this dimeric organization offers a possible mechanism for the ability of LARGE1 to synthesize long matriglycan chains. The structural information further reveals the mechanisms in which disease-causing mutations disrupt the activity of LARGE1. Collectively, these data shed light on how matriglycan is synthesized alongside the functional significance of glycosyltransferase oligomerization.

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“…Although the vast majority of OGT substrates are in IDRs (5), there are a few examples of proteins that are glycosylated in folded or ordered regions, including HBGB-1(74), H2B(75) and αB-Crystallin (76). Glycosylation of these ordered sites could occur if the TPR is able to move away from the catalytic site, as has been suggested by a recent electron microscopy structure (77). Alternatively, the OGT-TPR might be able to unfold a select group of ordered regions, as the TPR-containing karyopherin proteins are known to behave as chaperones (78).…”

Section: Substrate Structure Impact On Glycosylationmentioning

confidence: 92%

Exploration of O-GlcNAc-transferase (OGT) glycosylation sites reveals a target sequence compositional bias

Chong

Nosella

Vanama

et al. 2022

Preprint

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O-GlcNAc transferase (OGT) is an essential glycosylating enzyme that catalyzes the addition of N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. The enzyme glycosylates a broad range of peptide sequences and prediction of glycosylation sites has proven challenging. The lack of an experimentally verified set of polypeptide sequences that are not glycosylated by OGT has made prediction of legitimate glycosylation sites more difficult. Here, we tested a number of intrinsically disordered protein regions as substrates of OGT to establish a set of sequences that are not glycosylated by OGT. The negative data set suggests an amino acid compositional bias for OGT targets. This compositional bias was validated by modifying the amino acid composition of the protein Fused in sarcoma (FUS) to enhance glycosylation. NMR experiments demonstrate that the tetratricopeptide repeat (TPR) region of OGT can bind FUS and that glycosylation-promoting mutations enhance binding. These results provide evidence that the TPR recognizes disordered segments of substrates with particular compositions to promote glycosylation, providing insight into the broad specificity of OGT.

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Cryo-EM structure provides insights into the dimer arrangement of the O-linked β-N-acetylglucosamine transferase OGT

Cited by 28 publications

References 37 publications

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

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

Structural basis for matriglycan synthesis by the LARGE1 dual glycosyltransferase

Exploration of O-GlcNAc-transferase (OGT) glycosylation sites reveals a target sequence compositional bias

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