N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structural information. Here we present crystal structures of human GPT in complex with tunicamycin. Structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.
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Data for this study were collected at beamlines NECAT 24-ID-C and 24-ID-E and at SERCAT 22-ID, both at the Advanced Photon Source. We thank B. Chung for initial GPT biochemistry. We thank I. Tickle and the STARANISO team for help with our anisotropic data analysis. This work was supported by the National Institutes of Health (R01GM120594 and R35NS097241 to S.-Y. L.), JSPS Grant-in-Aid for Scientific Research (B) (16H05097 to S.I.), Astellas Foundation for Research on Metabolic Disorders (to S.I.), Hokkaido University GFC, PSOU, funded by MEXT (to S.I.), and BINDS from the Japan Agency for Medical Research and Development (to S.I.). Beamlines 24-ID-C and 24-ID-E are funded by P41 GM103403 and S10 RR029205.
The authors declare no competing financial interests.
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Integrated supplementary information
Supplementary Figure 1 Chemical structures of tunicamycin and the natural substrates of GPT and MraY.
Chemical structure of the competitive inhibitor tunicamycin A1 is shown at the top, while the structures of the natural substrates (UDP-GlcNAc for GPT and UDP-MurNAc-pentapeptide for MraY) are shown at the bottom. All compounds contain uridine moieties and either a GlcNAc or MurNAc moiety.
Supplementary Figure 2 The His129 and Pro129 variants of hGPT are active and have comparable specific activity.
a, TLC plate image of hGPT His129- and Pro129-catalyzed reactions (in triplicate) containing 200 nM of each enzyme, 500 μM C55-dol-P, 0.1 mM UDP-GlcNAc and 0.01 mM [14C]UDP-GlcNAc, 70 mM Tris-HCl pH 8.0, 500 mM NaCl, 80 mM MgCl2, 5 mM DM, 1 mg/mL POPG, and 10% glycerol. A time course assay was conducted at 30°C and 2 μL of each reaction was spotted on the TLC plate every 5 min; the 15 min time point shown is within the linear range. b, Specific activity measurements based on spot intensity quantification of the substrate and product bands shown in panel a. Three technical replicates are shown, with the mean value represented by a line.
2F o −F c composite omit maps were calculated from the canonical hGPT Pro129 data (3.1 Å resolution) omitting 5% of the model at a time, and contoured to 0.8 σ. a, hGPT dimer, showing ribbons for protomer A only. Omit density for protomer B is colored pink for clarity. b, View of the cytosolic cavity, showing tunicamycin and several nearby residues. c, View of the dimer interface from within the membrane, looking at protomer A from protomer B. d, View of the dimer interface from the cytosol. e, 2F o −F c composite omit map, shown for individual transmembrane helices 1 to 10 and carved 1.8 Å from the model.
The canonical hGPT (Pro129) is shown at the top (3.1 Å resolution), while the His129 variant is shown below (2.95 Å resolution). Tunicamycin is denoted as magenta sticks. 2F o –F c omit density of tunicamycin is shown in blue mesh (contoured to 0.8 σ), while the F o –F c omit density of tunicamycin is shown in green mesh (contoured to 3 σ). Maps were carved 1.8 Å from the tunicamycin model.
The sequences of human GPT, MraYAA (5CKR), and MraYCB (5JNQ) were first aligned by structural superposition, expanded to 45 GPT ortholog sequences by MAFFT, and corrected manually. Invariant positions (100% identity) are shaded red, while conserved positions (>90% identity) are shaded gray. The secondary structure of human GPT is shown, highlighting the insertion in loop E that is absent in MraY sequences (TM, transmembrane α-helix; α, α-helix; β, β-strand).
Omit maps were calculated with data of the canonical hGPT (Pro129) to 3.1 Å resolution. One lipid tail of 1-palmitoyl-2-oleoylglycero-3-phosphoglycerol (POPG, magenta sticks) is found in each side fenestration. 2F o –F c omit density of POPG molecules is shown in blue mesh (contoured to 0.8 σ), while the F o –F c omit density is shown in green mesh (contoured to 3 σ). Maps were carved 1.8 Å from the POPG molecules.
Positions of CDG1J-associated mutants are colored yellow, while positions of CMS13-associated mutants are colored blue.
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Yoo, J., Mashalidis, E.H., Kuk, A.C.Y. et al. GlcNAc-1-P-transferase–tunicamycin complex structure reveals basis for inhibition of N-glycosylation. Nat Struct Mol Biol 25, 217–224 (2018). https://doi.org/10.1038/s41594-018-0031-y
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