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Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase

Abstract

Oligosaccharyltransferase (OST) is a membrane-integral enzyme that catalyzes the transfer of glycans from lipid-linked oligosaccharides (LLOs) onto asparagine side chains, the first step in protein N-glycosylation. Here, we report the X-ray structure of a single-subunit OST, PglB from Campylobacter lari, trapped in an intermediate state bound to an acceptor peptide and a synthetic LLO analog. The structure reveals the role of the external loop EL5, present in all OST enzymes, in substrate recognition. Whereas the N-terminal half of EL5 binds LLO, the C-terminal half interacts with the acceptor peptide. The glycan moiety of LLO must thread under EL5 to access the active site. Reducing EL5 mobility decreases the catalytic rate of OST when full-size heptasaccharide LLO is provided, but not for a monosaccharide-containing LLO analog. Our results define the chemistry of a ternary complex state, assign functional roles to conserved OST motifs, and provide opportunities for glycoengineering by rational design of PglB.

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Figure 1: In vitro inhibition of PglB with synthetic LLO analogs.
Figure 2: Structure of the ternary complex.
Figure 3: LLO-binding site.
Figure 4: Effect of disaccharide linkage on PglB activity.
Figure 5: Disulfide cross-linking of EL5 to the TM domain of PglB.
Figure 6: Glycosylation mechanism.

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Acknowledgements

This research was supported by the Swiss National Science Foundation (SNF 310030B_166672 to K.P.L. and Transglyco Sinergia grant to M.A., J.-L.R. and K.P.L.). We thank the beamline staff at the Swiss Light Source for assistance with data collection, C. Lizak for preparing a cysteineless construct of PglB, and A. Ramírez for assistance with the chemo-enzymatic synthesis of farnesyl-PP-GlcNAc-1,3α-GalNAc.

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Authors

Contributions

M.N. performed the overexpression, purification, disulfide cross-linking, functional characterization, and crystallization of PglB. K.P.L. and M.N. performed X-ray data collection, structure determination, and model building and refinement. J.B., T.S., T.D., and J.-L.R. synthesized LLO analogs. M.N., M.A., and K.P.L. devised experiments and analyzed the data. K.P.L. and M.N. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Kaspar P Locher.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 In vitro activity of PglB with synthetic LLO analogs.

PglB activity was measured in vitro using synthetic LLO analogs with distinct polyprenyl tails. (a) Structures of synthetic LLO analogs featuring cis (ωZ-PP-GlcNAc) or trans (ωE-PP-GlcNAc) configuration of double bonds. (b) Tricine SDS-PAGE analysis of peptide glycosylation determined by quantification of fluorescently labeled substrate. The assays were performed at 30 °C and contained 1 μM PglB and 100 μM synthetic LLO analog. (c) Turnover numbers of glycosylation assays shown in b. Data represent three independent protein preparations (error bars indicate s.d., n=3).

Supplementary Figure 2 Structures of lipid-linked oligosaccharides.

(a) Structure of full-length, wild type C. jejuni LLO, abbreviated as undecaprenyl-PP-BacGalNAc5Glc. (b) Structure of synthetic, inhibitory LLO nerylneryl-PPC-GlcNAc as used for co-crystallization and structure determiation of PglB ternary complex.

Supplementary Figure 3 Kinetic analysis of synthetic LLO substrates.

Normalized PglB activities as a function of varying concentrations of three LLO analogs (structures shown in Fig. 1a). Activities were normalized to the maximal turnover rate for each LLO analog independently to allow the comparison of the Michaelis constants KM. Each data point represents three independent cell cultures, calculated from the slope of the linear regression fit (error bars indicate s.d., n≥4)

Supplementary Figure 4 PglB membrane topology.

Topological scheme of PglB transmembrane domain, with TM helices depicted as cylinders and numbered. Dashed green lines indicate non-covalent contacts to the periplasmic domain. The N-terminal and C-terminal segments of the external loop EL5 are indicated as N-EL5 and C-EL5, respectively.

Supplementary Figure 5 Surface representation of the ternary complex structure. PglB- acceptor peptide-LLO complex.

(a) Surface representation of the ternary complex structure of PglB bound to acceptor peptide and synthetic, inhibitory LLO analog. PglB is colored grey, N-EL5 in turquoise, C-EL5 in purple, bound peptide in orange, bound LLO in yellow. (b) Close-up view of a. The acceptor peptide and inhibitory LLO are shown in orange and yellow sticks, respectively. The substrates are bound to cavities at opposite entrances / exits of the PglB tunnel.

Supplementary Figure 6 Catalytic site and acceptor asparagine binding.

(a) Stereo view of the catalytic site and acceptor peptide binding. PglB is colored as in Fig. 2a, selected residues are shown as sticks and labeled. The acceptor peptide is shown as orange sticks, with residues numbered relative to their position in the acceptor sequon (Asn at position zero). The bound Mn2+ ion is shown as a pink sphere and labeled. (b) Schematic view of coordination geometry of Mn2+ ion, yellow dashed lines depict octahedral shape. The six ligands are the side-chain atoms of D56, D154, E319 and three water molecules, shown as red spheres.

Supplementary Figure 7 Disulfide cross-linking efficiency of the cysteine double mutant.

The ratio of disulfide cross-linking was determined by labeling unreacted, free cysteines with fluorescein maleimide and quantitation of in-gel fluorescence. (a) Coomassie-stained gel; (b) fluorescence-scanned gel. The oxidative disulfide bond formation was determined to be 80 +/- 2.9% from three independent cell cultures (error denotes s.d., n=3). Lanes labeled “ox” (oxidized) indicates a sample that was cross-linked with CuCl2 during the purification process, whereas “red” (reduced) indicates a control sample that contained 10 mM β-mercaptoethanol during purification, which was removed by desalting. MW denotes marker proteins, with masses indicated on the side.

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Uncropped gel image for Fig 4b (PDF 133 kb)

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Napiórkowska, M., Boilevin, J., Sovdat, T. et al. Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase. Nat Struct Mol Biol 24, 1100–1106 (2017). https://doi.org/10.1038/nsmb.3491

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