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Structural basis of protein arginine rhamnosylation by glycosyltransferase EarP

Nature Chemical Biology volume 14, pages 368–374 (2018)Cite this article

Abstract

Protein glycosylation regulates many cellular processes. Numerous glycosyltransferases with broad substrate specificities have been structurally characterized. A novel inverting glycosyltransferase, EarP, specifically transfers rhamnose from dTDP-β-l-rhamnose to Arg32 of bacterial translation elongation factor P (EF-P) to activate its function. Here we report a crystallographic study of Neisseria meningitidis EarP. The EarP structure contains two tandem Rossmann-fold domains, which classifies EarP in glycosyltransferase superfamily B. In contrast to other structurally characterized protein glycosyltransferases, EarP binds the entire β-sheet structure of EF-P domain I through numerous interactions that specifically recognize its conserved residues. Thus Arg32 is properly located at the active site, and causes structural change in a conserved dTDP-β-l-rhamnose-binding loop of EarP. Rhamnosylation by EarP should occur via an SN2 reaction, with Asp20 as the general base. The Arg32 binding and accompanying structural change of EarP may induce a change in the rhamnose-ring conformation suitable for the reaction.

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Fig. 1: Structures of EarP.
Fig. 2: EF-P recognition by EarP.
Fig. 3: Mutational analyses.
Fig. 4: Binding of dTDP-β-l-rhamnose by EarP.
Fig. 5: Possible conformational change of the rhamnose ring induced by EF-P binding.

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Acknowledgements

We thank the staff of beamlines BL32XU and BL26B2 at SPring-8 (Harima, Japan) and beamline BL5A at the Photon Factory (Tsukuba, Japan). We thank T. Terada, T. Imada, and T. Nakayama (RIKEN) for clerical assistance. We thank K. Ohtsuki, M. Usui, and A. Abe (RIKEN) for mass spectrometry analysis, and M. Kuratani and N. Sakai (RIKEN) for technical assistance. This work was supported by the Platform Project for Supporting Drug Discovery and Life Science Research from the Japan Agency for Medical Research and Development (AMED) (to S.Y.), and by JSPS KAKENHI (grant number 16K05859 to T.Y.). This research used the computational resources of the supercomputer HOKUSAI (RIKEN Advanced Center for Computing and Communications).

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Authors and Affiliations

  1. RIKEN Structural Biology Laboratory, Yokohama, Japan

    Toru Sengoku, Yasushi Hikida, Shigeyuki Yokoyama & Tatsuo Yanagisawa

  2. Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Wako, Japan

    Takehiro Suzuki & Naoshi Dohmae

  3. Structure-Based Molecular Design Team, RIKEN Center for Life Science Technologies, Yokohama, Japan

    Chiduru Watanabe & Teruki Honma

  4. Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN–Max Planck Joint Research Center, RIKEN Global Research Cluster, Wako, Japan

    Yoshiki Yamaguchi

  5. Department of Bacteriology, National Institute of Infectious Disease, Tokyo, Japan

    Hideyuki Takahashi

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Contributions

S.Y., H.T., and T.Y. conceived the project. T. Sengoku, S.Y., and T.Y. designed the experiments. T. Sengoku and T.Y. prepared protein samples. T. Sengoku crystallized proteins and performed crystallographic experiments and analyses. Y.H. assisted with the crystallographic analyses. N.D., T. Suzuki, and T.Y. performed mass spectrometry experiments. T.Y. performed ITC experiments. T. Sengoku, C.W., and T.H. performed the MD simulation, with advice on the sugar conformation from Y.Y. All authors analyzed the data. T. Sengoku, S.Y., and T.Y. wrote the manuscript.

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Correspondence to Shigeyuki Yokoyama or Tatsuo Yanagisawa.

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Sengoku, T., Suzuki, T., Dohmae, N. et al. Structural basis of protein arginine rhamnosylation by glycosyltransferase EarP. Nat Chem Biol 14, 368–374 (2018). https://doi.org/10.1038/s41589-018-0002-y

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