Structural basis of the molecular ruler mechanism of a bacterial glycosyltransferase

Ramírez, Ana S.; Boilevin, Jérémy; Mehdipour, Ahmad Reza; Hummer, Gerhard; Darbre, Tamis; Reymond, Jean‐Louis; Locher, Kapspar P.

doi:10.1038/s41467-018-02880-2

Cited by 32 publications

(42 citation statements)

References 59 publications

(81 reference statements)

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“…PglH is intriguing as it adds three saccharide units in a processive fashion and reactions occur distal to the membrane surface. In this case, the predicted placement of PglH in the membrane calculated using PPM server [49] reveals how the tilt of the structure relative to the membrane surface may accommodate these reactions by placing the active site cleft further from the membrane surface [50] compared to WbnH (Figure 4A). Insight into how PglH transfers predominantly three carbohydrate units comes from both mechanistic and structural analyses.…”

Section: A Marriage Of Conveniencementioning

confidence: 99%

“…Kinetic analysis with native substrates shows that each progressive sugar addition is retarded due to product inhibition, ultimately disfavoring further elongation of the product [51]. Additionally, recent structural analysis with substrates and substrate analogs suggests that specific interactions between the diphosphate moiety of the Pren-PP-glycan and basic residues on an α-helix at the membrane surface acts as a molecular ruler that forms the basis for substrate specificity and determines the final product composition [50]. Ultimately, the placement of the extended polyprenol moiety of the substrate in the membrane, married to the distal active-site binding of the soluble glycan moiety, takes full advantage of the monotopic enzyme topology of PglH.…”

Section: A Marriage Of Conveniencementioning

confidence: 99%

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Monotopic Membrane Proteins Join the Fold

Allen

Entova

Ray

et al. 2019

Trends in Biochemical Sciences

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Monotopic membrane proteins, classified by topology, are proteins which embed into a single face of the membrane. These proteins are generally underrepresented in the protein data bank, but the last decade of research has revealed new examples that allow description of generalizable features. This review summarizes shared characteristics including oligomerization states, modes of membrane association, mechanisms of interactions with hydrophobic or amphiphilic substrates and homology to soluble folds. We also discuss how association of monotopic enzymes into pathways can be used to promote substrate specificity and product composition. These examples highlight the challenges in structure determination specific to this class of proteins, but also the promise of new understanding from future study of these proteins that reside at the interface.

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Section: A Marriage Of Conveniencementioning

confidence: 99%

Section: A Marriage Of Conveniencementioning

confidence: 99%

Monotopic Membrane Proteins Join the Fold

Allen

Entova

Ray

et al. 2019

Trends in Biochemical Sciences

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“…Both enzymes possess a G instead FIGURE 2 | Sequence alignment of C. fetus (Cf) PglX, PglY, and C. jejuni (Cj) PglH. Black boxes indicate specific amino acids associated with activity in PglH (Troutman and Imperiali, 2009;Ramirez et al, 2018). Black and dark gray amino acids represent functional residues that show non-conserved substitutions in PglX and PglY.…”

Section: Characterization Of Cff Pgl Clustermentioning

confidence: 99%

Influence of Protein Glycosylation on Campylobacter fetus Physiology

et al. 2020

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Campylobacter fetus is commonly associated with venereal disease and abortions in cattle and sheep, and can also cause intestinal or systemic infections in humans that are immunocompromised, elderly, or exposed to infected livestock. It is also believed that C. fetus infection can result from the consumption or handling of contaminated food products, but C. fetus is rarely detected in food since isolation methods are not suited for its detection and the physiology of the organism makes culturing difficult. In the related species, Campylobacter jejuni, the ability to colonize the host has been linked to N-linked protein glycosylation with quantitative proteomics demonstrating that glycosylation is interconnected with cell physiology. Using label-free quantitative (LFQ) proteomics, we found more than 100 proteins significantly altered in expression in two C. fetus subsp. fetus protein glycosylation (pgl) mutants (pglX and pglJ) compared to the wild-type. Significant increases in the expression of the (NiFe)-hydrogenase HynABC, catalyzing H 2-oxidation for energy harvesting, correlated with significantly increased levels of cellular nickel, improved growth in H 2 and increased hydrogenase activity, suggesting that N-glycosylation in C. fetus is involved in regulating the HynABC hydrogenase and nickel homeostasis. To further elucidate the function of the C. fetus pgl pathway and its enzymes, heterologous expression in Escherichia coli followed by mutational and functional analyses revealed that PglX and PglY are novel glycosyltransferases involved in extending the C. fetus hexasaccharide beyond the conserved core, while PglJ and PglA have similar activities to their homologs in C. jejuni. In addition, the pgl mutants displayed decreased motility and ethidium bromide efflux and showed an increased sensitivity to antibiotics. This work not only provides insight into the unique protein N-glycosylation pathway of C. fetus, but also expands our knowledge on the influence of protein N-glycosylation on Campylobacter cell physiology.

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“…Binding profiles were visualised using pyGenomeTracks [12]. Heatmaps were generated via the ComplexHeatmap R package [13].…”

Section: Bioinformatic Analysismentioning

confidence: 99%

Membrane-bound flurophore-labelled vectors for Targeted DamID allow simultaneous profiling of expression domains and DNA binding

Delandre

McMullen

Marshall

2020

Preprint

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Targeted DamID (TaDa) allows highly efficient celltype-specific profiling of protein-DNA interactions. Cell-type-specificity, however, is governed by the GAL4/UAS system, which can exhibit differences in expression patterns depending upon the genomic insertion site and the UAS promoter strength. The TaDa system uses a bicistronic transcript to reduce the translation rates of Dam-fusion proteins, presenting the possibility of using the primary ORF within in the transcript to label expression domains and precisely identified the profiled cell populations in experimental samples. Here, we describe two TaDa vectors, pTaDaG and pTaDaG2, that use myristoylated GFP as the primary ORF. Differing lengths of the myristoylation sequence between the plasmids allows additional translational control. Fly lines created with this system allow easy visualisation of expression domains under both fluorescent dissecting and confocal microscopes without the use of antibody staining, whilst faithfully profiling protein-DNA interactions via Targeted DamID.

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Structural basis of the molecular ruler mechanism of a bacterial glycosyltransferase

Cited by 32 publications

References 59 publications

Monotopic Membrane Proteins Join the Fold

Monotopic Membrane Proteins Join the Fold

Influence of Protein Glycosylation on Campylobacter fetus Physiology

Membrane-bound flurophore-labelled vectors for Targeted DamID allow simultaneous profiling of expression domains and DNA binding

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