GlcNAc-1-P-transferase–tunicamycin complex structure reveals basis for inhibition of N-glycosylation

Abstract

SummaryN-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, 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 by 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. Our 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 for the design of MraY-specific inhibitors as potential antibiotics.

Improvement of homologous GH10 xylanase production by deletion of genes with predicted function in the Aspergillus nidulans secretion pathway

“…S4), and thus short exposition periods were selected to perform RNA‐seq analysis (Appendix S1). Cellular stress was induced with Tm and DTT, which prevent N‐glycosylation of newly synthesized proteins and the formation of disulphide bonds, respectively (Fan et al , 2018; Yoo et al , 2018). A total of 294 million reads were generated, more than 94% of the filtered reads were mapped to the A. nidulans FGSC A4 reference genome, and a total of 10,774 genes were analysed (Table S1).…”

Structures of DPAGT1 Explain Glycosylation Disease Mechanisms and Advance TB Antibiotic Design

“…Five loops connect the TMHs on the cytoplasmic side of the membrane (CL1, -3, -5, -7, and -9), which form the active site, 3 loops on the ER side of the membrane (EL2, -4, -6) and one (EL8) embedded in the membrane on the ER side. DPAGT1 has a similar overall fold to MraY (Chung et al, 2013;Yoo et al, 2018) ( Figure 1E). A feature of the eukaryotic DPAGT1 PNPT family not found in prokaryotic PNPTs is a 52-residue insertion between Arg306-Cys358 in CL9, following TMH9.…”

“…Together these results confirmed our hypothesis that systematic ''lipid alteration'' could create tunicamycin analogues in which mammalian cytotoxicity is separated from antibacterial effects. A Molecular Explanation for Differences in TUN-X,X Analogue Binding to DPAGT1 and MraY Comparison of the structures of the complexes of tunicamycin with DPAGT1 and MraY (Hakulinen et al, 2017;Yoo et al, 2018; this work) gave an explanation for selectivity of analogues on MraY over DPAGT1. The MraY tunicamycin binding site has a more open, shallow surface than in DPAGT1; in the latter the lipid tail is completely enclosed by Trp122 adjacent to the active site ( Figure 6G).…”

“…The protein production methods, structures, assays and complexes are components of a ''target enabling package'' developed at the Structural Genomics Consortium (released June 2017, http://www.thesgc. org/tep/DPAGT1), which has already been used by others (Yoo et al, 2018). These structures, combined with mutagenesis and activity analysis, reveal both the mechanism of catalysis by DPAGT1 and the molecular basis of DPAGT1related diseases.…”

Visualizing Conformation Transitions of the Lipid Flippase MurJ

“…To accomplish this task, cells utilize a translocase to attach the oligosaccharide precursor to a lipid carrier [1][2][3] , and a flippase to transport the lipid-linked oligosaccharide (LLO) across the membrane 4 . Such LLO transport systems are evolutionarily ancient and found across all domains of life from bacteria to humans 5,6 .…”