Structure and mechanism of the ER-based glucosyltransferase ALG6

Bloch, J.S.; Pesciullesi, Giorgio; Boilevin, Jérémy; Nosol, Kamil; Irobalieva, Rossitza N.; Darbre, Tamis; Aebi, Markus; Kossiakoff, Anthony A.; Reymond, Jean‐Louis; Locher, Kaspar P.

doi:10.1038/s41586-020-2044-z

Cited by 62 publications

(55 citation statements)

References 60 publications

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“…Because no lipids were added during purification, these phospholipids are potentially endogenous substrates that were co-purified with the Dnf1-Lem3 complex. This is not unusual, because previous studies have shown that substrate lipids bound in the substrate pockets can be co-purified with the enzyme complexes (Bai et al, 2019b;Bloch et al, 2020;Hiraizumi et al, 2019). Interestingly, the three phospholipids are out of register with respect to the lipid bilayer by approximately 10 Å: PL1 is moved down about 10 Å into the middle of the bilayer relative to lipids in the outer leaflet, and PL2 and PL3 are moved 10 Å into the cytosolic space relative to lipids in the inner leaflet of the membrane.…”

Section: Phospholipids Bind At the Exoplasmic And Cytoplasmic Sites Omentioning

confidence: 76%

Transport Mechanism of Class-3 P4 ATPase Lipid Flippases

Bai

You

Jain

et al. 2020

Preprint

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The P4 ATPases are a family of membrane enzymes that transport lipids across membrane bilayers. The structures of the class-1 phosphatidylserine flippases have been reported, revealing a substrate site on lumenal side. However, the cytosolic binding site has not been found, and the transport mechanisms of P4 ATPases in other classes are unknown. Here we report a structural and functional study on plasma-membrane localized, class-3 P4 ATPases Dnf1–Lem3 and Dnf2–Lem3. We captured substrate lipids on both the exoplasmic and cytosolic sides, and found that these two enzymes have similar structures. We found a helix-turn-helix motif in the cytosolic P domain that is unique to the class-3 enzymes and plays a crucial role in their function. Unexpectedly, Lem3 contributes to substrate binding near the cytosolic surface. Conformational transitions of these two class-3 enzymes match those of the class-1 enzymes, suggesting a conserved mechanism among all classes of P4 ATPases.

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Section: Phospholipids Bind At the Exoplasmic And Cytoplasmic Sites Omentioning

confidence: 76%

Transport Mechanism of Class-3 P4 ATPase Lipid Flippases

Bai

You

Jain

et al. 2020

Preprint

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“…Here, we decided to validate both the transformer and the augmented precision of the CARBO model on a recently realized synthetic sequence from our own laboratory, absent from the training data. This sequence is a 14-step synthesis of lipid-linked oligosaccharide (LLO) 15 to be used as a substrate to study OST 46 , 47 (Fig. 4 ).…”

Section: Resultsmentioning

confidence: 99%

Transfer learning enables the molecular transformer to predict regio- and stereoselective reactions on carbohydrates

et al. 2020

Self Cite

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Organic synthesis methodology enables the synthesis of complex molecules and materials used in all fields of science and technology and represents a vast body of accumulated knowledge optimally suited for deep learning. While most organic reactions involve distinct functional groups and can readily be learned by deep learning models and chemists alike, regio- and stereoselective transformations are more challenging because their outcome also depends on functional group surroundings. Here, we challenge the Molecular Transformer model to predict reactions on carbohydrates where regio- and stereoselectivity are notoriously difficult to predict. We show that transfer learning of the general patent reaction model with a small set of carbohydrate reactions produces a specialized model returning predictions for carbohydrate reactions with remarkable accuracy. We validate these predictions experimentally with the synthesis of a lipid-linked oligosaccharide involving regioselective protections and stereoselective glycosylations. The transfer learning approach should be applicable to any reaction class of interest.

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“…Based on the published structures of known GT‐C fold glycosyltransferases, GT‐C enzymes are composed of structurally conserved N‐terminal module and variable C‐terminus (Bloch et al ., 2020). The conserved N‐terminal modules consisting the first seven transmembrane helices interact with the dolichol moiety (Bloch et al ., 2020). The first external loop contains a conserved EXD, DXE, DD, or DE motif that is essential for catalysis (Lommel et al ., 2011).…”

Section: Discussionmentioning

confidence: 99%

“…The first external loop contains a conserved EXD, DXE, DD, or DE motif that is essential for catalysis (Lommel et al ., 2011). The C‐terminal modules of different GT‐C enzymes vary in fold and topology, and interact with diverse mono or oligosaccharides attached to the dolichol carrier (Bloch et al ., 2020). The loop 1 in the N‐terminus of Agl22 contains a DE motif; however, it was predicted in the inner side of the membrane by the CCTOP server (Figure 1a).…”

Section: Discussionmentioning

confidence: 99%

Agl22 and Agl23 are involved in the synthesis and utilization of the lipid‐linked intermediates in the glycosylation pathways of the halophilic archaeaon Haloarcula hispanica

Lü

Pei

Zhou

et al. 2020

Molecular Microbiology

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Like both eukaryotes and bacteria, archaea can decorate proteins with N‐ and O‐linked glycans. Whereas pathways and roles of N‐glycosylation have been studied in several model archaeal organisms, little is known of O‐glycosylation. To explore commonalities and variations of these two versions of glycosylation, we used Haloarcula hispanica as a model. Our previous work showed that H. hispanica S‐layer glycoproteins are modified by an N‐linked glucose‐α‐(1, 2)‐[sulfoquinovosamine‐β‐(1, 6)‐]galactose trisaccharide and an O‐linked glucose‐α‐(1, 4)‐galactose disaccharide. Here, we found that H. hispanica membrane contains C60 dolichol phosphate (DolP) as a lipid carrier for glycosylation. As revealed by bioinformatics, gene deletion and phenotype analysis, gene HAH_1571, renamed agl22, encodes a predicted glucosyltransferase that transfers glucose from glucose‐DolP onto galactose‐DolP to form the glucose‐α‐(1, 4)‐galactose‐DolP precursor of the N‐glycosylation. Gene HAH_2016, renamed agl23, encodes a putative flippase‐associated protein responsible for flipping of hexose‐DolPs across the membrane to face the exterior. Our results also suggested that the synthesis of the N‐ and O‐linked glycans onto target protein occurs on the outer surface of the cell using hexose‐DolPs as sugar donors. Deletion mutant showed that N‐ and O‐glycosylation are required for growth in the defined medium mimicking the natural habitat of H. hispanica.

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Structure and mechanism of the ER-based glucosyltransferase ALG6

Cited by 62 publications

References 60 publications

Transport Mechanism of Class-3 P4 ATPase Lipid Flippases

Transport Mechanism of Class-3 P4 ATPase Lipid Flippases

Transfer learning enables the molecular transformer to predict regio- and stereoselective reactions on carbohydrates

Agl22 and Agl23 are involved in the synthesis and utilization of the lipid‐linked intermediates in the glycosylation pathways of the halophilic archaeaon Haloarcula hispanica

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