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Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase

Nature Chemical Biology volume 13, pages 1261–1266 (2017)Cite this article

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Abstract

Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion using noncanonical amino acids, yet its structure and function are not completely understood. Here we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNAPyl recognition by PylRS is anticodon independent: the anticodon does not contact the enzyme. Then, using standard microbiological culture equipment, we established a new method for laboratory evolution—a noncontinuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. Finally, we employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a noncanonical amino acid.

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Figure 1: Crystal structures of the wild-type and PANCE-evolved PylRS variants bound to tRNAPyl.
Figure 2: Structural basis for the PylRS specificity to tRNAPyl.
Figure 3: PANCE method application and overview.

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Acknowledgements

The authors thank S. Melnikov and Y. Xiong (Yale University) for insightful discussions and intellectual contributions, A. Shinoda (Paul Scherrer Institute) and K. Yamashita (RIKEN) for advice on structure analysis, and S. Trauger (Small Molecule Mass Spectrometry Laboratory at Harvard University) for providing expertise with intact protein mass spectrometry analysis. This work was supported the US National Institutes of Health (NIH) R01EB022376 and R35GM118062 (to D.R.L.), and R01GM022854 and R35GM122560 (to D.S.), by the Defense Advanced Research Projects Agency N66001-12-C-4207 (to D.R.L.), by the Department of Energy DE-FG02-98ER20311 (to D.S.), and the Howard Hughes Medical Institute. D.I.B is supported by the National Institutes of Health under a Ruth L. Kirschstein National Research Service Award (F32GM106621). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.

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Author notes

  1. Corwin Miller

    Present address: DS Therapeutics, Houston, Texas, USA

  2. Yane-Shih Wang

    Present address: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

  3. Tateki Suzuki and Corwin Miller: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Tateki Suzuki, Corwin Miller, Li-Tao Guo, Joanne M L Ho, Yane-Shih Wang & Dieter Söll

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    David I Bryson & David R Liu

  3. Howard Hughes Medical Institute, Cambridge, Massachusetts, USA

    David R Liu

  4. Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA

    David R Liu

  5. Department of Chemistry, Yale University, New Haven, Connecticut, USA

    Dieter Söll

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Contributions

T.S. purified and crystallized the PylRS–tRNAPyl complexes, solved structures, and analyzed data. C.M. designed the PANCE research, performed experiments, analyzed data, and wrote the manuscript. D.S. designed and supervised the research and wrote the manuscript. D.R.L. designed and supervised the research and edited the manuscript. L.-T.G. designed the chimeric chPylRS variant for evolution in PANCE, performed protein purification and in vitro aminoacylation assays, and analyzed data. J.M.L.H. assisted with design and refinement of PANCE procedure. D.I.B. established initial selection conditions, performed read-through assays, and performed mass spectrometry and western blot analyses. All authors contributed to editing the manuscript.

Corresponding author

Correspondence to Dieter Söll.

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Suzuki, T., Miller, C., Guo, LT. et al. Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase. Nat Chem Biol 13, 1261–1266 (2017). https://doi.org/10.1038/nchembio.2497

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