Search

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the Salmonella enterica (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs., Competing Interests: GD, AA, YH, WL, RV, BI No competing interests declared, (© 2023, Dodge et al.)

Complex bacterial glycoconjugates drive interactions between pathogens, symbionts, and their human hosts. Glycoconjugate biosynthesis is initiated at the membrane interface by phosphoglycosyl transferases (PGTs), which catalyze the transfer of a phosphosugar from a soluble uridine diphosphosugar (UDP-sugar) substrate to a membrane-bound polyprenol-phosphate (Pren-P). The two distinct superfamilies of PGT enzymes (polytopic and monotopic) show striking differences in their structure and mechanism. We designed and synthesized a series of uridine bisphosphonates (UBPs), wherein the diphosphate of the UDP and UDP-sugar is replaced by a substituted methylene bisphosphonate (CXY-BPs; X/Y = F/F, Cl/Cl, ( S )-H/F, ( R )-H/F, H/H, CH 3 /CH 3 ). UBPs and UBPs incorporating an N -acetylglucosamine (GlcNAc) substituent at the β-phosphonate were evaluated as inhibitors of a polytopic PGT (WecA from Thermotoga maritima ) and a monotopic PGT (PglC from Campylobacter jejuni ). Although CHF-BP most closely mimics diphosphate with respect to its acid/base properties, the less basic CF 2 -BP conjugate more strongly inhibited PglC, whereas the more basic CH 2 -BP analogue was the strongest inhibitor of WecA. These surprising differences indicate different modes of ligand binding for the different PGT superfamilies, implicating a modified P-O - interaction with the structural Mg 2+ . For the monoPGT enzyme, the two diastereomeric CHF-BP conjugates, which feature a chiral center at the P α -CHF-P β carbon, also exhibited strikingly different binding affinities and the inclusion of GlcNAc with the native α-anomer configuration significantly improved binding affinity. UBP-sugars are thus revealed as informative new mechanistic probes of PGTs that may aid development of novel antibiotic agents for the exclusively prokaryotic monoPGT superfamily.

Complex glycans serve essential functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane-associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked substrates of specific isoprene unit geometry, such as undecaprenol phosphate (UndP) in bacteria. How these enzymes and substrates interact within a lipid bilayer needs further investigation. Here, we focus on a small enzyme, PglC from Campylobacter, structurally characterized for the first time in 2018 as a detergent-solubilized construct. PglC is a monotopic phosphoglycosyl transferase that embodies the functional core structure of the entire enzyme superfamily and catalyzes the first membrane-committed step in a glycoprotein assembly pathway. The size of the enzyme is significant as it enables high-level computation and relatively facile, for a membrane protein, experimental analysis. Our ensemble computational and experimental results provided a high-level view of the membrane-embedded PglC/UndP complex. The findings suggested that it is advantageous for the polyprenol phosphate to adopt a conformation in the same leaflet where the monotopic membrane protein resides as opposed to additionally disrupting the opposing leaflet of the bilayer. Further, the analysis showed that electrostatic steering acts as a major driving force contributing to the recognition and binding of both UndP and the soluble nucleotide sugar substrate. Iterative computational and experimental mutagenesis support a specific interaction of UndP with phosphoglycosyl transferase cationic residues and suggest a role for critical conformational transitions in substrate binding and specificity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Phosphoglycosyl transferases (PGTs) are among the first membrane-bound enzymes involved in the biosynthesis of bacterial glycoconjugates. Robust expression and purification protocols for an abundant subfamily of PGTs remains lacking. Recent advancements in detergent-free methods for membrane protein solubilization open the door for purification of difficult membrane proteins directly from cell membranes into native-like liponanoparticles. By leveraging autoinduction, in vivo SUMO tag cleavage, styrene maleic acid co-polymer liponanoparticles (SMALPs), and Strep-Tag purification, we have established a robust workflow for expression and purification of previously unobtainable PGTs. The material generated from this workflow is extremely pure and can be directly visualized by Cryogenic Electron Microscopy (CryoEM). The methods presented here promise to be generalizable to additional membrane proteins recombinantly expressed in E. coli and should be of interest to the greater membrane proteomics community., Competing Interests: Declaration of competing interest The authors declare that they have no competing personal or financial relationships that could appear to influence the work reported herein., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Monotopic phosphoglycosyl transferases (monoPGTs) are an expansive superfamily of enzymes that catalyze the first membrane-committed step in the biosynthesis of bacterial glycoconjugates. MonoPGTs show a strong preference for their cognate nucleotide diphospho-sugar (NDP-sugar) substrates. However, despite extensive characterization of the monoPGT superfamily through previous development of a sequence similarity network comprising >38,000 nonredundant sequences, the connection between monoPGT sequence and NDP-sugar substrate specificity has remained elusive. In this work, we structurally characterize the C-terminus of a prototypic monoPGT for the first time and show that 19 C-terminal residues play a significant structural role in a subset of monoPGTs. This new structural information facilitated the identification of co-conserved sequence "fingerprints" that predict NDP-sugar substrate specificity for this subset of monoPGTs. A Hidden Markov model was generated that correctly assigned the substrate of previously unannotated monoPGTs. Together, these structural, sequence, and biochemical analyses have delivered new insight into the determinants guiding substrate specificity of monoPGTs and have provided a strategy for assigning the NDP-sugar substrate of a subset of enzymes in the superfamily that use UDP-di-N-acetyl bacillosamine. Moving forward, this approach may be applied to identify additional sequence motifs that serve as fingerprints for monoPGTs of differing UDP-sugar substrate specificity., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Monotopic phosphoglycosyl transferase enzymes (monoPGTs) initiate the assembly of prokaryotic glycoconjugates essential for bacterial survival and proliferation. MonoPGTs belong to an expansive superfamily with a diverse and richly annotated sequence space; however, the biochemical roles of most monoPGTs in glycoconjugate biosynthesis pathways remain elusive. To better understand these critical enzymes, we have implemented activity-based protein profiling (ABPP) probes as protein-centric, membrane protein compatible tools that lay the groundwork for understanding the activity and regulation of the monoPGT superfamily from a cellular proteome. With straightforward gel-based readouts, we demonstrate robust, covalent labeling at the active site of various representative monoPGTs from cell membrane fractions using 3-phenyl-2H-azirine probes.

Phosphoglycosyl transferases (PGTs) are integral membrane proteins with diverse architectures that catalyze the formation of polyprenol diphosphate-linked glycans via phosphosugar transfer from a nucleotide diphosphate-sugar to a polyprenol phosphate. There are two PGT superfamilies that differ significantly in overall structure and topology. The polytopic PGT superfamily, represented by MraY and WecA, has been the subject of many studies because of its roles in peptidoglycan and O-antigen biosynthesis. In contrast, less is known about a second, extensive superfamily of PGTs that reveals a core structure with dual domain architecture featuring a C-terminal soluble globular domain and a predicted N-terminal membrane-associated domain. Representative members of this superfamily are the Campylobacter PglCs, which initiate N-linked glycoprotein biosynthesis and are implicated in virulence and pathogenicity. Despite the prevalence of dual domain PGTs, their mechanism of action is unknown. Here, we present the mechanistic analysis of PglC, a prototypic dual domain PGT from Campylobacter concisus Using a luminescence-based assay, together with substrate labeling and kinetics-based approaches, complementary experiments were carried out that support a ping-pong mechanism involving a covalent phosphosugar intermediate for PglC. Significantly, mass spectrometry-based approaches identified Asp93, which is part of a highly conserved AspGlu dyad found in all dual domain PGTs, as the active-site nucleophile of the enzyme involved in the formation of the covalent adduct. The existence of a covalent phosphosugar intermediate provides strong support for a ping-pong mechanism of PglC, differing fundamentally from the ternary complex mechanisms of representative polytopic PGTs., Competing Interests: The authors declare no conflict of interest.

Oligosaccharyl transferase is part of the macromolecular machinery that processes nascent proteins in the endoplasmic reticulum. The enzyme is highly conserved, catalyzes the initial step in the biosynthesis of N-linked glycoproteins and acts as a 'gatekeeper' for the secretory pathway. As more proteins associated with oligosaccharyl transferase are identified, the intricacies of the enzyme and the relationship with other proteins in the lumen of the endoplasmic reticulum are starting to be unraveled.

Linear hexapeptides featuring the asparagine mimetics alanine-beta-hydrazide, alanine-beta-hydroxylamine, and 1,3-diaminobutanoic acid have been synthesized as oligosaccharyl transferase (OT) substrate mimetics and chemoselectively N-glycosylated to obtain the corresponding neoglycopeptides as OT product mimetics. The effect of glycosylation on the binding of these asparagine surrogates is in stark contrast with the effect of modification of native asparagine. In native N-linked glycosylation, product inhibition is minimal and glycopeptides show very low affinity for OT. In contrast, glycosylation of the substrate mimetics maintains or even improves affinity of the corresponding product mimetic for OT. Conformational considerations suggest that the flexibility of the N-glycosyl linkage in these neoglycopeptides allows them to be accommodated in the OT binding site while the native trans glycosyl amide linkage is rejected. These results provide insight into how OT minimizes product inhibition, thereby ensuring effective substrate turnover.

Oligosaccharyl transferase (OT) catalyzes the co-translational transfer of a dolichol-linked tetradecasaccharide (Dol-PP-GlcNAc(2)Man(9)Glc(3), 1a) to an asparagine side chain of a nascent polypeptide inside the lumen of the endoplasmic reticulum (ER). The glycosyl acceptor requires an Asn-Xaa-Thr/Ser sequon, where Xaa can be any natural amino acid except proline, for N-linked glycosylation to occur. To address the substrate specificity of the glycosyl donor, three unnatural dolichol-linked disaccharide analogues (Dol-PP-GlcNTFA-GlcNAc 1c, Dol-PP-2DFGlc-GlcNAc 1d, and Dol-PP-GlcNAc-Glc 1e) were synthesized and evaluated as substrates or inhibitors for OT from yeast. The synthetic analogue Dol-PP-GlcNAc-Glc 1e, with substitution in the distal sugar, was found to be a substrate (K(m)(app)() = 26 microM) for OT. On the other hand, the analogues Dol-PP-GlcNTFA-GlcNAc 1c (K(i) = 154 microM) and Dol-PP-2DFGlc-GlcNAc 1d (K(i) = 252 microM), with variations in the proximal sugar, were inhibitors for OT. The dolichol-linked monosaccharide Dol-PP-GlcNAc 3 was found to be the minimum unit for glycosylation to occur.

Protein glycosylation is associated with many critical biological processes. In connection with studies on the mechanism of asparagine-linked glycosylation by the enzyme oligosaccharyl transferase, we have prepared peptide inhibitors that interact with the enzyme at nanomolar concentrations. Herein we describe efforts directed toward improving the binding properties of these inhibitors.

Recent work has resulted in the development of potent inhibitors of oligosaccharyl transferase (OT), the enzyme that catalyzes the cotranslational glycosylation of asparagine [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Soc. 118, 7636-7637; Kellenberger, C., Hendrickson, T. L., and Imperiali, B. (1997) Biochemistry 36, 12554-12559]. However, no specific OT inhibitors that function in the cellular environment have yet been reported. The peptide cyclo(hex-Amb-Cys)-Thr-Val-Thr-Nph-NH2 was previously shown to exhibit nanomolar inhibition (Ki = 37 nM) through slow tight binding kinetics [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Soc. 118, 7636-7637]. Included herein is the redesign of this prototype inhibitor for achieving both passive and active translocation into model membrane systems representing the endoplasmic reticulum (ER). The strategy for passive transport involved the incorporation of a membrane permeable import function previously shown to carry various peptides across the outer as well as the interior cellular membranes [Rojas, M., Donahue, J. P., Tan, Z., and Lin, Y.-Z. (1998) Nat. Biotechnol. 16, 370-375]. Assessment of function in intact ER membranes revealed that the inhibitor targeted toward passive diffusion demonstrated concentration-dependent inhibition of two different glycosylation substrates. Thus, this modified inhibitor achieved potent inhibition of glycosylation after being successfully transported through the ER membrane. In the active translocation approach, the lead OT inhibitor and a corresponding substrate were redesigned to include features recognized by the transporter associated with antigen processing (TAP). This protein translocates peptides into the lumen of the ER [Heemels, M.-T., Schumacher, T. N. M., Wonigeit, K., and Ploegh, H. L. (1993) Science 262, 2059-2063]. However, although acceptance of the cyclized substrate by the TAP receptor was demonstrated via efficient transport and glycosylation, the modified inhibitor was not translocated by TAP machinery, and therefore, active translocation was achieved for the modified substrate only. Both of these ER transport methods afforded redesigned OT inhibitors that retained their inhibitor properties in vitro, regardless of the extensions to the carboxy-terminus of the root inhibitor. The above family of redesigned inhibitors provides a template for generating a transcellular pathway and represents the first step toward OT inhibition in intact cells.

The peptide cyclo(hex-Amb(1)-Cys(2))-Thr(3)-Val(4)-Thr(5)-Nph(6)-NH2 was previously shown to be a slow, tight-binding inhibitor (Ki = 37 nM) of the yeast oligosaccharyl transferase (OT) [Hendrickson et al. (1996) J. Am. Chem. Soc. 118, 7636-7637]. This enzyme catalyzes the transfer of a carbohydrate moiety to an asparagine residue in the consensus sequence Asn-Xaa-Thr/Ser. Herein we present a study of the contribution of the residues in positions 1, 3, 4, and 5 to OT binding. Replacement of the threonine (residue 3) by valine or (S)-2-aminobutyric acid dramatically reduced the potency of the inhibitor while, surprisingly, the incorporation of an additional methylene into the side chain of residue 1 [(S)-2,3-diaminobutyric acid changed to ornithine] had very little effect. Variants with acidic, basic, hydrophilic/polar, and hydrophobic side chains in positions 4 and 5 were also evaluated for both yeast and porcine liver OT inhibition. This aspect of the study reveals that basic (lysine) and acidic (glutamic acid) residues are detrimental to the binding, whereas hydrophobic (valine) and polar/hydrophilic (threonine) residues are both well tolerated. The kinetic behavior of substrate analogs [cyclo(hex-Asn(1)-Cys(2))-Thr(3)-Xaa(4)-Yaa(5)-Nph-NH2] corresponding to inhibitors of weak, medium, and strong potency was also examined in order to provide insight into the nature of these inhibitors.

Oligosaccharyl transferase catalyzes the glycosylation of selected asparagine residues of nascent polypeptide chains as they are translocated into the lumen of the endoplasmic reticulum. To date, this enzyme has been purified from a number of eukaryotic organisms. Purification of transferase activity has yielded polypeptide complexes of three to six subunits depending on the source organism. Here we present the purification of an affinity-tagged version of the enzyme complex from a membrane protein fraction of the yeast Saccharomyces cerevisiae. A yeast strain was created in which the essential 64-kDa glycoprotein Nlt1p subunit of the oligosaccharyl transferase was modified by the addition of a 22-residue carboxy-terminal affinity tag; the tag included both an 8-residue FLAG epitope and a 6-residue histidine motif. Facile purification of the oligosaccharyl transferase was achieved using affinity chromatography media specific for each segment of the tag. The enzyme was purified as a heteromeric complex of five subunits in agreement with previously reported characterizations of the yeast transferase. Yeast strains bearing affinity-tagged enzyme subunits allow the rapid characterization of native and mutant transferase complexes.

Oligosaccharyl transferase activity exhibits an absolute requirement for certain divalent metal cations. Studies with reconstituted enzyme suggest a preference for metal ions that can adopt an octahedral coordination geometry. In order to gain insight into the specific role of the metal cation in catalysis, we have investigated the influence of the metal cofactor on catalytic turnover of the tripeptide substrate Bz-Asn-Leu-Thr-NHMe (1) and a closely related sulfur-containing analog, Bz-Asn(gamma S)-Leu-Thr-NHMe (2). The metal ion substitution studies reveal that 1 is effectively turned over in the presence of several metal ions (Mn2+, Fe2+, Mg2+, and Ca2+). In contrast, 2 is only glycosylated in the presence of the thiophilic metal cations manganese and iron. When the enzyme is reconstituted with the oxophilic cations magnesium and calcium, 2 shows minimal substrate behavior. With the amide substrate 1, the distinct preference for manganese over magnesium may argue against direct coordination of the metal to the lipid-linked substrate pyrophosphate moiety. This fact, together with the comparative studies with asparagine- and thioasparagine-containing tripeptides, implicates the metal cofactor in a role that places it proximal to the peptide binding site.

Chemical labeling of the multimeric Saccharomyces cerevisiae oligosaccharyl transferase indicates that the 48 kDa Wbp1p subunit is an integral component of the catalytically active enzyme. The enzyme was purified following chromatography on concanavalin A agarose, heparin agarose, Q-Sepharose, and hydroxyapatite media. The enzyme activity copurified with a tetrameric complex of polypeptide subunits. Two of the subunits have been identified as the yeast proteins Wbp1p and Swp1p by amino-terminal residue sequencing. A third subunit was identified as a variably glycosylated polypeptide near 64 kDa; preliminary amino acid sequencing showed no identity to known yeast proteins. Modification of a cysteine residue by the reagent methyl methanethiolsulfonate (MMTS) caused time-dependent and concentration-dependent inactivation of the enzyme. To identify the modified subunit of the transferase complex, the labeling reagent S-[(N-biotinoylamino)ethyl] methanethiolsulfonate (BMTS) was synthesized. Like MMTS, BMTS inactivated the oligosaccharyl transferase in a time-dependent manner. Additionally, incubation with the substrate (dolichylpyrophosphoryl)-N,N'-diacetylchitobiose [Dol-PP(GlcNAc)2] protected the enzyme from BMTS inactivation. When the purified enzyme complex was incubated with BMTS, Wbp1p alone was specifically labeled, thereby associating this subunit with catalysis and the binding of the dolichylpyrophosphoryl oligosaccharide substrate in the transferase reaction.

The yeast oligosaccharyl transferase catalyzes the glycosylation of asparagine residues in secreted, vesicular, and membrane proteins. A complex of at least four membrane-bound polypeptides is responsible for oligosaccharyl transferase activity. Amino acid sequences from the 64 kDa glycoprotein subunit of the complex were used to clone the essential NLT1 (N-linked oligosaccharyl transferase) gene. The Nlt1p gene product is a processed, multiply glycosylated type I membrane protein; it has an extensive amino-terminal soluble domain, a potential hydrophobic transmembrane domain, and a short carboxy-terminal soluble domain. The Nlt1p is significantly similar than the mammalian ribophorin I, a component of the mammalian oligosaccharyl transferase complex, and the enzyme is conserved throughout eukaryotic evolution.

A series of tripeptides that satisfy the -Asn-Xaa-Thr/Ser- primary sequence requirement [Marshall, R. D. (1972) Annu. Rev. Biochem. 41, 673-702] for N-glycosylation have been synthesized and examined as potential acceptors in an oligosaccharyltransferase assay. Of these, six (Ac-Asn-Ala-Thr-NH2, Ac-Asn-Leu-Thr-NH2, Ac-Asn-Asp-Thr-NH2, Ac-Asn-D-Ala-Thr-NH2, Ac-Asn-Pro-Thr-NH2, and Ac-Asn-AIB-Thr-NH2) were examined for solution conformational properties in dimethyl sulfoxide with use of amide proton temperature coefficients, 3JHN alpha analysis [Pardi, A., et al. (1984) J. Mol. Biol. 180, 741-751], and 2-D ROESY experiments [Bothner-By, A. A., et al. (1984) J. Am. Chem. Soc. 106, 811-813]. The analysis reveals that the peptides that serve as acceptors in the transferase assay demonstrate similar conformational properties in solution. These are highlighted by a secondary structural motif that involves the interaction between the asparagine side-chain carboxamide and the backbone amide of the threonine. The peptides that show very poor acceptor, or even nonacceptor, properties in the oligosaccharyltransferase assay demonstrate different conformational features in solution. These observations may explain the distinct biological activity observed for these peptides.