Synthesis of rhamnosylated arginine glycopeptides and determination of the glycosidic linkage in bacterial elongation factor P

Wang, Siyao; Corcilius, Leo; Sharp, Phillip P.; Rajkovic, Andrei; Ibba, Michael; Parker, Benjamin L.; Payne, Richard J.

doi:10.1039/c6sc03847f

Cited by 26 publications

(30 citation statements)

References 31 publications

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“…1B and supplemental Annotations). Although glycosylation events are typically labile under collision-based fragmentation, we observed Arg-GlcNAc to be partially stable, consistent with previous observations of Arg rhamnosylation (29,30). This stability enabled the localization of Arg-GlcNAc to the previously reported sites for FADD (Arg 117 ; Fig.…”

Section: Development Of An Arg-glcnac Immunoenrichment Methodssupporting

confidence: 90%

The bacterial arginine glycosyltransferase effector NleB preferentially modifies Fas-associated death domain protein (FADD)

Scott

Giogha

Pollock

et al. 2017

Journal of Biological Chemistry

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The inhibition of host innate immunity pathways is essential for the persistence of attaching and effacing pathogens such as enteropathogenic (EPEC) and during mammalian infections. To subvert these pathways and suppress the antimicrobial response, attaching and effacing pathogens use type III secretion systems to introduce effectors targeting key signaling pathways in host cells. One such effector is the arginine glycosyltransferase NleB1 (NleB in ) that modifies conserved arginine residues in death domain-containing host proteins with-acetylglucosamine (GlcNAc), thereby blocking extrinsic apoptosis signaling. Ectopically expressed NleB1 modifies the host proteins Fas-associated via death domain (FADD), TNFRSF1A-associated via death domain (TRADD), and receptor-interacting serine/threonine protein kinase 1 (RIPK1). However, the full repertoire of arginine GlcNAcylation induced by pathogen-delivered NleB1 is unknown. Using an affinity proteomic approach for measuring arginine-GlcNAcylated glycopeptides, we assessed the global profile of arginine GlcNAcylation during ectopic expression of NleB1, EPEC infection , or infection NleB overexpression resulted in arginine GlcNAcylation of multiple host proteins. However, NleB delivery during EPEC and infection caused rapid and preferential modification of Arg in FADD. This FADD modification was extremely stable and insensitive to physiological temperatures, glycosidases, or host cell degradation. Despite its stability and effect on the inhibition of apoptosis, arginine GlcNAcylation did not elicit any proteomic changes, even in response to prolonged NleB1 expression. We conclude that, at normal levels of expression during bacterial infection, NleB1/NleB antagonizes death receptor-induced apoptosis of infected cells by modifying FADD in an irreversible manner.

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Section: Development Of An Arg-glcnac Immunoenrichment Methodssupporting

confidence: 90%

The bacterial arginine glycosyltransferase effector NleB preferentially modifies Fas-associated death domain protein (FADD)

Scott

Giogha

Pollock

et al. 2017

Journal of Biological Chemistry

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“…The rhamnosylation of Arg32* of EF-P was shown to be performed by EarP, a conserved glycosyltransferase encoded at a position adjacent to efp and employing dTDP-␤-L-rhamnose (TDP-Rha) as the donor substrate (7,21,22). Li et al and Wang et al reported that rhamnose is ␣-linked to Arg32* in bacterial EF-P proteins, demonstrating that EarP inverts the sugar of its donor substrate (27,28). During the preparation of our manuscript, two independent works on the structure of EarP appeared.…”

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confidence: 99%

Complex Structure of Pseudomonas aeruginosa Arginine Rhamnosyltransferase EarP with Its Acceptor Elongation Factor P

Liu

et al. 2019

J Bacteriol

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A bacterial inverting glycosyltransferase EarP transfers rhamnose from dTDP-β-l-rhamnose (TDP-Rha) to Arg32 of translation elongation factor P (EF-P) to activate its function. We report here the structural and biochemical characterization of Pseudomonas aeruginosa EarP. In contrast to recently reported Neisseria meningitidis EarP, P. aeruginosa EarP exhibits differential conformational changes upon TDP-Rha and EF-P binding. Sugar donor binding enhances acceptor binding to EarP, as revealed by structural comparison between the apo-, TDP-Rha-, and TDP/EF-P-bound forms and isothermal titration calorimetry experiments. In vitro EF-P rhamnosylation combined with active-site geometry indicates that Asp16 corresponding to Asp20 of N. meningitidis EarP is the catalytic base, whereas Glu272 is another putative catalytic residue. Our study should provide the basis for EarP-targeted inhibitor design against infections from P. aeruginosa and other clinically relevant species. IMPORTANCE Posttranslational rhamnosylation of EF-P plays a key role in Pseudomonas aeruginosa, establishing virulence and antibiotic resistance, as well as survival. The detailed structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP from P. aeruginosa not only demonstrates that sugar donor TDP-Rha binding enhances acceptor EF-P binding to EarP but also should provide valuable information for the structure-guided development of its inhibitors against infections from P. aeruginosa and other EarP-containing pathogens.

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“…We and others previously showed that EarP inverts the anomeric configuration on the sugar moiety from TDP-β-L-rhamnose to α-rhamnosyl arginine. [24][25] Reportedly, inverting glycosyltransferases employ a direct displacement SN2-like reaction. 46 The molecular basis for inverted N-linked glycosylation was elucidated for the oligosaccharyl transferase PglB.…”

Section: D13 D17 and E273 Are Involved In Catalysis To Activate The mentioning

confidence: 99%

“…[18][19][20][21] By contrast, activation of a phylogenetically distinct group of EF-Ps encoded in species such as Pseudomonas aeruginosa, or Neisseria meningitidis, depends on rhamnosylation of an arginine R32 EF-P in the equivalent position. 15,[22][23] Rhamnosylation is mediated by the recently discovered glycosyltransferase EarP, which inverts the rhamnosyl moiety of the donor nucleotide sugar dTDP-β-L-rhamnose (TDP-Rha) into α-rhamnosyl-arginine when attached to EF-P. [24][25] Compared to the common and relatively well understood asparagine glycosylation, sugar modifications on the guanidino group of arginine appeared to be rare and almost nothing is known about the molecular mechanism. [26][27] Beside EF-P arginine-glycosylation, to date there are only two further reported cases: The first one described self β-glycosylation of sweet corn amylogenin.…”

mentioning

confidence: 99%

Structural basis for EarP-mediated arginine glycosylation of translation elongation factor EF-P

Krafczyk

Macošek²,

Gast

et al. 2017

Preprint

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15Glycosylation is a universal strategy to post-translationally modify proteins. The recently discovered 16 arginine rhamnosylation activates the polyproline specific bacterial translation elongation factor EF-P. 17 EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic 18 mechanism remains elusive. In the present study, we solved the crystal structure of EarP from 19Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann-folds, thus 20 constituting a GT-B glycosyltransferase. While TDP-rhamnose is located within a highly conserved 21 pocket of the C-domain, EarP recognizes EF-P via its KOW-like N-domain. Based on our structural 22 data combined with an in vitro / in vivo enzyme characterization, we propose a mechanism of 23 inverting arginine glycosylation. As EarP is essential for pathogenicity in P. aeruginosa our study 24 provides the basis for targeted inhibitor design. 25

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Synthesis of rhamnosylated arginine glycopeptides and determination of the glycosidic linkage in bacterial elongation factor P

Abstract: We describe the synthesis and incorporation of α- and β-configured rhamnosyl arginine cassettes into Pseudomonas aeruginosa elongation factor P-derived glycopeptides. These were used to unequivocally determine the native anomeric configuration of the rhamnose moiety in EF-P.

Cited by 26 publications

References 31 publications

The bacterial arginine glycosyltransferase effector NleB preferentially modifies Fas-associated death domain protein (FADD)

The bacterial arginine glycosyltransferase effector NleB preferentially modifies Fas-associated death domain protein (FADD)

Complex Structure of Pseudomonas aeruginosa Arginine Rhamnosyltransferase EarP with Its Acceptor Elongation Factor P

Structural basis for EarP-mediated arginine glycosylation of translation elongation factor EF-P

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