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.

  • Article
  • Published:

Structural dynamics of the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase

Nature Structural & Molecular Biology volume 19, pages 677–684 (2012)Cite this article

Subjects

Abstract

Leucyl-tRNA synthetase (LeuRS) produces error-free leucyl-tRNALeu by coordinating translocation of the 3′ end of (mis-)charged tRNAs from its synthetic site to a separate proofreading site for editing. Here we report cocrystal structures of the Escherichia coli LeuRS–tRNALeu complex in the aminoacylation or editing conformations, showing that translocation involves correlated rotations of four flexibly linked LeuRS domains. This pivots the tRNA to guide its charged 3′ end from the closed aminoacylation state to the editing site. The editing domain unexpectedly stabilizes the tRNA during aminoacylation, and a large rotation of the leucine-specific domain positions the conserved KMSKS loop to bind the 3′ end of the tRNA, promoting catalysis. Our results give new insight into the structural dynamics of a molecular machine that is essential for accurate protein synthesis.

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: Structures of the E. coli LeuRS–tRNALeu complex in the aminoacylation and editing states.
Figure 2: LeuRS–tRNALeu interactions in the aminoacylation complex.
Figure 3: Comparison between the aminoacylation and the editing configurations.
Figure 4: Binding of the tRNA 3′ end induces full closure of the KMSKS loop.
Figure 5: Dynamics of the functional cycle of bacterial LeuRS.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Karkhanis, V.A., Mascarenhas, A.P. & Martinis, S.A. Amino acid toxicities of Escherichia coli that are prevented by leucyl-tRNA synthetase amino acid editing. J. Bacteriol. 189, 8765–8768 (2007).

    Article  CAS  Google Scholar 

  2. Lincecum, T.L. Jr. et al. Structural and mechanistic basis of pre- and posttransfer editing by leucyl-tRNA synthetase. Mol. Cell 11, 951–963 (2003).

    Article  CAS  Google Scholar 

  3. Tukalo, M., Yaremchuk, A., Fukunaga, R., Yokoyama, S. & Cusack, S. The crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer-editing conformation. Nat. Struct. Mol. Biol. 12, 923–930 (2005).

    Article  CAS  Google Scholar 

  4. Hagiwara, Y., Field, M.J., Nureki, O. & Tateno, M. Editing mechanism of aminoacyl-tRNA synthetases operates by a hybrid ribozyme/protein catalyst. J. Am. Chem. Soc. 132, 2751–2758 (2010).

    Article  CAS  Google Scholar 

  5. Rock, F.L. et al. An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site. Science 316, 1759–1761 (2007).

    Article  CAS  Google Scholar 

  6. Seiradake, E. et al. Crystal structures of the human and fungal cytosolic leucyl-tRNA synthetase editing domains: A structural basis for the rational design of antifungal benzoxaboroles. J. Mol. Biol. 390, 196–207 (2009).

    Article  CAS  Google Scholar 

  7. Zhang, Y.K. et al. Synthesis and structure-activity relationships of novel benzoxaboroles as a new class of antimalarial agents. Bioorg. Med. Chem. Lett. 21, 644–651 (2011).

    Article  CAS  Google Scholar 

  8. Ding, D. et al. Design, synthesis, and structure-activity relationship of Trypanosoma brucei leucyl-tRNA synthetase inhibitors as antitrypanosomal agents. J. Med. Chem. 54, 1276–1287 (2011).

    Article  CAS  Google Scholar 

  9. Fukai, S. et al. Structural basis for double-sieve discrimination of L-valine from L-isoleucine and L-threonine by the complex of tRNAVal and valyl-tRNA synthetase. Cell 103, 793–803 (2000).

    Article  CAS  Google Scholar 

  10. Silvian, L.F., Wang, J. & Steitz, T.A. Insights into editing from an ile-tRNA synthetase structure with tRNAIle and mupirocin. Science 285, 1074–1077 (1999).

    Article  CAS  Google Scholar 

  11. Fukunaga, R. & Yokoyama, S. Aminoacylation complex structures of leucyl-tRNA synthetase and tRNALeu reveal two modes of discriminator-base recognition. Nat. Struct. Mol. Biol. 12, 915–922 (2005).

    Article  CAS  Google Scholar 

  12. Cusack, S., Yaremchuk, A. & Tukalo, M. The 2 crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue. EMBO J. 19, 2351–2361 (2000).

    Article  CAS  Google Scholar 

  13. Fukunaga, R. & Yokoyama, S. Crystal structure of leucyl-tRNA synthetase from the archaeon Pyrococcus horikoshii reveals a novel editing domain orientation. J. Mol. Biol. 346, 57–71 (2005).

    Article  CAS  Google Scholar 

  14. Perona, J.J., Rould, M.A. & Steitz, T.A. Structural basis for transfer RNA aminoacylation by Escherichia coli glutaminyl-tRNA synthetase. Biochemistry 32, 8758–8771 (1993).

    Article  CAS  Google Scholar 

  15. Rath, V.L., Silvian, L.F., Beijer, B., Sproat, B.S. & Steitz, T.A. How glutaminyl-tRNA synthetase selects glutamine. Structure 6, 439–449 (1998).

    Article  CAS  Google Scholar 

  16. Bullock, T.L., Uter, N., Nissan, T.A. & Perona, J.J. Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants. J. Mol. Biol. 328, 395–408 (2003).

    Article  CAS  Google Scholar 

  17. Sekine, S. et al. ATP binding by glutamyl-tRNA synthetase is switched to the productive mode by tRNA binding. EMBO J. 22, 676–688 (2003).

    Article  CAS  Google Scholar 

  18. Sekine, S. et al. Structural bases of transfer RNA-dependent amino acid recognition and activation by glutamyl-tRNA synthetase. Structure 14, 1791–1799 (2006).

    Article  CAS  Google Scholar 

  19. Yaremchuk, A., Kriklivyi, I., Tukalo, M. & Cusack, S. Class I tyrosyl-tRNA synthetase has a class II mode of cognate tRNA recognition. EMBO J. 21, 3829–3840 (2002).

    Article  CAS  Google Scholar 

  20. Kobayashi, T. et al. Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion. Nat. Struct. Biol. 10, 425–432 (2003).

    Article  CAS  Google Scholar 

  21. Yang, X.L. et al. Two conformations of a crystalline human tRNA synthetase-tRNA complex: implications for protein synthesis. EMBO J. 25, 2919–2929 (2006).

    Article  CAS  Google Scholar 

  22. Shen, N. et al. Catalytic mechanism of the tryptophan activation reaction revealed by crystal structures of human tryptophanyl-tRNA synthetase in different enzymatic states. Nucleic Acids Res. 36, 1288–1299 (2008).

    Article  CAS  Google Scholar 

  23. Hauenstein, S., Zhang, C.M., Hou, Y.M. & Perona, J.J. Shape-selective RNA recognition by cysteinyl-tRNA synthetase. Nat. Struct. Mol. Biol. 11, 1134–1141 (2004).

    Article  CAS  Google Scholar 

  24. Konno, M. et al. Modeling of tRNA-assisted mechanism of Arg activation based on a structure of Arg-tRNA synthetase, tRNA, and an ATP analog (ANP). FEBS J. 276, 4763–4779 (2009).

    Article  CAS  Google Scholar 

  25. Delagoutte, B., Moras, D. & Cavarelli, J. tRNA aminoacylation by arginyl-tRNA synthetase: induced conformations during substrates binding. EMBO J. 19, 5599–5610 (2000).

    Article  CAS  Google Scholar 

  26. Tocchini-Valentini, G., Saks, M.E. & Abelson, J. tRNA leucine identity and recognition sets. J. Mol. Biol. 298, 779–793 (2000).

    Article  CAS  Google Scholar 

  27. Larkin, D.C., Williams, A.M., Martinis, S.A. & Fox, G.E. Identification of essential domains for Escherichia coli tRNALeu aminoacylation and amino acid editing using minimalist RNA molecules. Nucleic Acids Res. 30, 2103–2113 (2002).

    Article  CAS  Google Scholar 

  28. Crepin, T. et al. Use of analogues of methionine and methionyl adenylate to sample conformational changes during catalysis in Escherichia coli methionyl-tRNA synthetase. J. Mol. Biol. 332, 59–72 (2003).

    Article  CAS  Google Scholar 

  29. Li, T., Guo, N., Xia, X., Wang, E.D. & Wang, Y.L. The peptide bond between E292–A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity. Biochemistry 38, 13063–13069 (1999).

    Article  CAS  Google Scholar 

  30. Du, X. & Wang, E.D. E292 is important for the aminoacylation activity of Escherichia coli leucyl-tRNA synthetase. J. Protein Chem. 22, 71–76 (2003).

    Article  CAS  Google Scholar 

  31. Lue, S.W. & Kelley, S.O. An aminoacyl-tRNA synthetase with a defunct editing site. Biochemistry 44, 3010–3016 (2005).

    Article  CAS  Google Scholar 

  32. Kobayashi, T. et al. Structural snapshots of the KMSKS loop rearrangement for amino acid activation by bacterial tyrosyl-tRNA synthetase. J. Mol. Biol. 346, 105–117 (2005).

    Article  CAS  Google Scholar 

  33. Vu, M.T. & Martinis, S.A. A unique insert of leucyl-tRNA synthetase is required for aminoacylation and not amino acid editing. Biochemistry 46, 5170–5176 (2007).

    Article  CAS  Google Scholar 

  34. Reader, J.S. et al. Major biocontrol of plant tumors targets tRNA synthetase. Science 309, 1533 (2005).

    Article  CAS  Google Scholar 

  35. Li, L. et al. Naturally occurring aminoacyl-tRNA synthetases editing-domain mutations that cause mistranslation in Mycoplasma parasites. Proc. Natl. Acad. Sci. USA 108, 9378–9383 (2011).

    Article  CAS  Google Scholar 

  36. Uter, N.T. & Perona, J.J. Active-site assembly in glutaminyl-tRNA synthetase by tRNA-mediated induced fit. Biochemistry 45, 6858–6865 (2006).

    Article  CAS  Google Scholar 

  37. Weimer, K.M., Shane, B.L., Brunetto, M., Bhattacharyya, S. & Hati, S. Evolutionary basis for the coupled-domain motions in Thermus thermophilus leucyl-tRNA synthetase. J. Biol. Chem. 284, 10088–10099 (2009).

    Article  CAS  Google Scholar 

  38. Han, J.M. et al. Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway. Cell 149, 410–424 (2012).

    Article  CAS  Google Scholar 

  39. Bonfils, G. et al. Leucyl-tRNA synthetase controls TORC1 via the EGO complex. Mol. Cell 46, 105–110 (2012).

    Article  CAS  Google Scholar 

  40. Larkin, D.C., Williams, A.M., Martinis, S.A. & Fox, G.E. Identification of essential domains for Escherichia coli tRNALeu aminoacylation and amino acid editing using minimalist RNA molecules. Nucleic Acids Res. 30, 2103–2113 (2002).

    Article  CAS  Google Scholar 

  41. Vu, M.T. & Martinis, S.A. A unique insert of leucyl-tRNA synthetase is required for aminoacylation and not amino acid editing. Biochemistry 46, 5170–5176 (2007).

    Article  CAS  Google Scholar 

  42. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  43. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  44. McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C. & Read, R.J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol. Crystallogr. 61, 458–464 (2005).

    Article  Google Scholar 

  45. Cusack, S., Yaremchuk, A. & Tukalo, M. The 2 crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue. EMBO J. 19, 2351–2361 (2000).

    Article  CAS  Google Scholar 

  46. Liu, Y., Liao, J., Zhu, B., Wang, E.D. & Ding, J. Crystal structures of the editing domain of Escherichia coli leucyl-tRNA synthetase and its complexes with Met and Ile reveal a lock-and-key mechanism for amino acid discrimination. Biochem. J. 394, 399–407 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  48. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  49. Hayward, S. & Lee, R.A. Improvements in the analysis of domain motions in proteins from conformational change: DynDom version 1.50. J. Mol. Graph. Model. 21, 181–183 (2002).

    Article  CAS  Google Scholar 

  50. Chen, V.B. . et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  51. Lincecum, T.L. Jr. et al. Structural and mechanistic basis of pre- and posttransfer editing by leucyl-tRNA synthetase. Mol. Cell 11, 951–963 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank the ESRF-EMBL Joint Structural Biology Group for access to ESRF beamlines and the EMBL-ESRF-ILL-IBS Partnership for Structural Biology for access to structural biology instrumentation, notably the high-throughput crystallization platform.

Author information

Author notes

  1. Andrés Palencia and Thibaut Crépin: These authors contributed equally to this work.

Authors and Affiliations

  1. European Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions, University of Grenoble–EMBL–Centre National de la Recherche Scientifique, Grenoble, France

    Andrés Palencia, Thibaut Crépin & Stephen Cusack

  2. Department of Biochemistry, University of Illinois, Roger Adams Laboratory, Urbana, Illinois, USA

    Michael T Vu, Tommie L Lincecum Jr & Susan A Martinis

Authors
  1. Andrés Palencia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thibaut Crépin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael T Vu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tommie L Lincecum Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Susan A Martinis
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stephen Cusack
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.C. crystallized the editing-state complexes and A.P. crystallized the aminoacylation complex. T.C., A.P. and S.C. collected X-ray data and performed the structural analysis. M.T.V. and T.L.L. performed the mutagenesis and associated biochemical studies under the supervision of S.A.M. S.C. wrote the manuscript with input from all other authors. S.A.M. acknowledges funding from US National Institutes of Health grant GM63789.

Corresponding author

Correspondence to Stephen Cusack.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Table 1 (PDF 3589 kb)

Supplementary Video 1

The aminoacylation and proof-reading functional cycle of bacterial LeuRS. The animation shows the conformational changes associated with the transition between the aminoacylation and editing states of LeuRSEC. The tRNA initial configuration corresponds to the editing state (the preferred conformation for the tRNA 3′ end) as observed in the structure in the absence of the leucyl-adenylate analogue (placed at top right in white balls and sticks for carbon, and standard colours for the rest of atoms). After binding of the substrate the video shows successively (with each change repeated) the translocation of the tRNA 3′ end to the catalytic site, the associated domain conformational movements, the transfer of leucine to the tRNA (concomitant to the release of AMP, right bottom) followed by the translocation to the editing site for proof-reading, and finally the release of the correctly charged leucyl-tRNALeu. The colour code for the protein domains is as in main text Figure 1 and key catalytic residues are represented by cyan sticks. The tRNA is a blue ribbon with the last base, Ade76, represented as ball and sticks with a filled base. The video was generated by the program CHIMERA. (MOV 25922 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Palencia, A., Crépin, T., Vu, M. et al. Structural dynamics of the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase. Nat Struct Mol Biol 19, 677–684 (2012). https://doi.org/10.1038/nsmb.2317

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2317

This article is cited by

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