Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB–MoaD complex

Nature volume 414, pages 325–329 (2001)Cite this article

Abstract

The activation of ubiquitin and related protein modifiers1,2 is catalysed by members of the E1 enzyme family that use ATP for the covalent self-attachment of the modifiers to a conserved cysteine. The Escherichia coli proteins MoeB and MoaD are involved in molybdenum cofactor (Moco) biosynthesis, an evolutionarily conserved pathway3,4. The MoeB- and E1-catalysed reactions are mechanistically similar, and despite a lack of sequence similarity, MoaD and ubiquitin display the same fold including a conserved carboxy-terminal Gly-Gly motif5. Similar to the E1 enzymes, MoeB activates the C terminus of MoaD to form an acyl-adenylate. Subsequently, a sulphurtransferase converts the MoaD acyl-adenylate to a thiocarboxylate that acts as the sulphur donor during Moco biosynthesis6,7. These findings suggest that ubiquitin and E1 are derived from two ancestral genes closely related to moaD and moeB3,5. Here we present the crystal structures of the MoeB–MoaD complex in its apo, ATP-bound, and MoaD-adenylate forms, and highlight the functional similarities between the MoeB– and E1–substrate complexes. These structures provide a molecular framework for understanding the activation of ubiquitin, Rub, SUMO and the sulphur incorporation step during Moco and thiamine biosynthesis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure of the MoeB–MoaD complex.
Figure 2: MoeB-catalysed activation of MoaD.
Figure 3: Active site conservation in the MoeB/E1 enzyme superfamily.

Similar content being viewed by others

References

  1. Hochstrasser, M. All in the ubiquitin family. Science 289, 563–564 (2000).

    Article  CAS  Google Scholar 

  2. Hochstrasser, M. Evolution and function of ubiquitin-like protein-conjugation systems. Nature Cell Biol. 2, E153–E157 (2000).

    Article  CAS  Google Scholar 

  3. Rajagopalan, K. V. in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (ed. Neidhardt, F. C.) 674–679 (ASM Press, Washington DC, 1996).

    Google Scholar 

  4. Rajagopalan, K. V. Biosynthesis and processing of the molybdenum cofactors. Biochem. Soc. Trans. 25, 757–761 (1997).

    Article  CAS  Google Scholar 

  5. Rudolph, M. J., Wuebbens, M. M., Rajagopalan, K. V. & Schindelin, H. Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation. Nature Struct. Biol. 8, 42–46 (2001).

    Article  CAS  Google Scholar 

  6. Leimkühler, S., Wuebbens, M. M. & Rajagopalan, K. V. Characterization of Escherichia coli MoeB and its involvement in the activation of MPT synthase for the biosynthesis of the molybdenum cofactor. J. Biol. Chem. 276, 34695–34701 (2001).

    Article  Google Scholar 

  7. Pitterle, D. M., Johnson, J. L. & Rajagopalan, K. V. In vitro synthesis of molybdopterin from precursor Z using purified converting factor. J. Biol. Chem. 268, 13506–13509 (1993).

    CAS  PubMed  Google Scholar 

  8. Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945–951 (1982).

    Article  CAS  Google Scholar 

  9. Burch, T. J. & Haas, A. L. Site-directed mutagenesis of ubiquitin. Differential roles for arginine in the interaction with ubiquitin-activating enzyme. Biochemistry 33, 7300–7308 (1994).

    Article  CAS  Google Scholar 

  10. Arnez, J. G., Dock-Bregeon, A. C. & Moras, D. Glycyl-tRNA synthetase uses a negatively charged pit for specific recognition and activation of glycine. J. Mol. Biol. 286, 1449–1459 (1999).

    Article  CAS  Google Scholar 

  11. Hatfield, P. M. & Vierstra, R. D. Multiple forms of ubiquitin-activating enzyme E1 from wheat. J. Biol. Chem. 267, 14799–14803 (1992).

    CAS  PubMed  Google Scholar 

  12. Lauhon, C. T. & Kambampati, R. The iscS gene in Escherichia coli is required for the biosynthesis of 4-thiouridine, thiamin, and NAD. J. Biol. Chem. 275, 20096–20103 (2000).

    Article  CAS  Google Scholar 

  13. Palenchar, P. M., Buck, C. J., Cheng, H., Larson, T. J. & Mueller, E. G. Evidence that ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis, may be a sulfurtransferase that proceeds through a persulfide intermediate. J. Biol. Chem. 275, 8283–8286 (2000).

    Article  CAS  Google Scholar 

  14. Xi, J., Ge, Y., Kinsland, C., McLafferty, F. W. & Begley, T. P. Biosynthesis of the thiazole moiety of thiamin in Escherichia coli: identification of an acyldisulfide-linked protein-protein conjugate that is functionally analogous to the ubiquitin/E1 complex. Proc. Natl Acad. Sci. USA 98, 8513–8518 (2001).

    Article  ADS  CAS  Google Scholar 

  15. Leimkühler, S. & Rajagopalan, K. V. An Escherichia coli NifS-like sulfurtransferase is required for the transfer of cysteine sulfur in the in vitro synthesis of molybdopterin from precursor Z. J. Biol. Chem. 276, 22024–22031 (2001).

    Article  Google Scholar 

  16. Otwinowski, Z. & Minor, W. in Methods in Enzymology: Macromolecular Crystallography (eds Carter, C. W. & Sweet, R. M. ) 307–326 (Academic, San Diego, 1997).

    Book  Google Scholar 

  17. Sheldrick, G. M. & Schneider, T. R. in Methods in Enzymology: Macromolecular Crystallography (eds Carter, C. W. & Sweet, R. M.) 319–343 (Academic, San Diego, 1997).

    Book  Google Scholar 

  18. DeLaFortelle, E. & Bricogne, G. in Methods in Enzymology: Macromolecular Crystallography (eds Carter, C. W. & Sweet, R. M.) 472–494 (Academic, San Diego, 1997).

    Book  Google Scholar 

  19. Abrahams, J. P. & Leslie, A. G. W. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996).

    Article  CAS  Google Scholar 

  20. Perrakis, A., Morris, R. & Lamzin, V. S. Automated protein model building combined with iterative structure refinement. Nature Struct. Biol. 6, 458–463 (1999).

    Article  CAS  Google Scholar 

  21. Bailey, S. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

    Article  Google Scholar 

  22. Murshudov, G., Vagin, A. & Dodson, E. Refinement of macromolecular structures by the maximum likelihood method. Acta Crystallogr. D 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  23. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  24. Johnson, M. E. & Rajagopalan, K. V. In vitro system for molybdopterin biosynthesis. J. Bacteriol. 169, 110–116 (1987).

    Article  CAS  Google Scholar 

  25. Laskowski, R. A., Moss, D. S. & Thornton, J. M. Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231, 1049–1067 (1993).

    Article  CAS  Google Scholar 

  26. Kraulis, P. J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  27. Barton, G. J. ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng. 6, 37–40 (1993).

    Article  CAS  Google Scholar 

  28. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. J. Rudolph for initial help with crystallization and data collection, J. Daniels for technical assistance, and D. Schneider for support at beamline X26C. This work was supported by National Institutes of Health (NIH) grants to H.S. and K.V.R.. The National Synchrotron Light Source in Brookhaven is supported by DOE and NIH, and beamline X26C is supported in part by the State University of New York at Stony Brook and its Research Foundation.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Center for Structural Biology, State University of New York at Stony Brook, Stony Brook, 11794-5115, New York, USA

    Michael W. Lake & Hermann Schindelin

  2. Department of Biochemistry, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Margot M. Wuebbens & K. V. Rajagopalan

Authors
  1. Michael W. Lake
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Margot M. Wuebbens
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. V. Rajagopalan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hermann Schindelin
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lake, M., Wuebbens, M., Rajagopalan, K. et al. Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB–MoaD complex. Nature 414, 325–329 (2001). https://doi.org/10.1038/35104586

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35104586

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing