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Pyrrolysyl-tRNA synthetase–tRNAPyl structure reveals the molecular basis of orthogonality

Nature volume 457, pages 1163–1167 (2009)Cite this article

Abstract

Pyrrolysine (Pyl), the 22nd natural amino acid, is genetically encoded by UAG and inserted into proteins by the unique suppressor tRNAPyl (ref. 1). The Methanosarcinaceae produce Pyl and express Pyl-containing methyltransferases that allow growth on methylamines2. Homologous methyltransferases and the Pyl biosynthetic and coding machinery are also found in two bacterial species1,3. Pyl coding is maintained by pyrrolysyl-tRNA synthetase (PylRS), which catalyses the formation of Pyl-tRNAPyl (refs 4, 5). Pyl is not a recent addition to the genetic code. PylRS was already present in the last universal common ancestor6; it then persisted in organisms that utilize methylamines as energy sources. Recent protein engineering efforts added non-canonical amino acids to the genetic code7,8. This technology relies on the directed evolution of an ‘orthogonal’ tRNA synthetase–tRNA pair in which an engineered aminoacyl-tRNA synthetase (aaRS) specifically and exclusively acylates the orthogonal tRNA with a non-canonical amino acid. For Pyl the natural evolutionary process developed such a system some 3 billion years ago. When transformed into Escherichia coli, Methanosarcina barkeri PylRS and tRNAPyl function as an orthogonal pair in vivo5,9. Here we show that Desulfitobacterium hafniense PylRS–tRNAPyl is an orthogonal pair in vitro and in vivo, and present the crystal structure of this orthogonal pair. The ancient emergence of PylRS–tRNAPyl allowed the evolution of unique structural features in both the protein and the tRNA. These structural elements manifest an intricate, specialized aaRS–tRNA interaction surface that is highly distinct from those observed in any other known aaRS–tRNA complex; it is this general property that underlies the molecular basis of orthogonality.

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Figure 1: Overall structures.
Figure 2: Structural alignment of tRNA synthetase–tRNA complexes.
Figure 3: DhPylRS–tRNA Pyl interface.
Figure 4: Comparison of PylRS structures.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 2zni (DhPylRS–tRNAPyl complex) and 2znj (DhPylRS apo).

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Acknowledgements

We thank the beamline staff at BL41XU of SPring-8 (Harima, Japan) and NW12 of PF-AR (Tsukuba, Japan) for technical help during data collection. P.O. holds a National Science Foundation postdoctoral fellowship in Biological Informatics. This work was supported by grants from the Japan Science and Technology Agency (to O.N.), from the National Project on Protein Structural and Functional Analyses of the Ministry of Education, Culture, Sports, Science and Technology (to O.N.), from the Ministry of Education, Culture, Sports, Science and Technology (to R.I. and O.N.), from the Mitsubishi Foundation (to O.N.), from the Kurata Memorial Hitachi Science and Technology Foundation (to O.N.), from the National Institute of General Medical Sciences (to D.S.), from the Department of Energy (to D.S.), and from the National Science Foundation (to D.S.).

Author Contributions K.N. performed purification, crystallization and structure determination. S.G. and T.U. conducted biochemical analyses. R.I. performed molecular dynamics. Y.A., R.I. and O.N. assisted the structure determination. P.O’D. analysed the data and performed bioinformatic analysis. P.O’D., K.N., O.N. and D.S. wrote the paper. O.N. and D.S. conceived and supervised the work.

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Author notes

  1. Kayo Nozawa, Patrick O’Donoghue and Sarath Gundllapalli: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B34 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan,

    Kayo Nozawa, Yuhei Araiso & Osamu Nureki

  2. Department of Molecular Biophysics and Biochemistry,,

    Patrick O’Donoghue, Sarath Gundllapalli, Takuya Umehara & Dieter Söll

  3. Department of Chemistry, Yale University, New Haven, Connecticut 06520-8114, USA,

    Dieter Söll

  4. Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan,

    Ryuichiro Ishitani & Osamu Nureki

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  1. Kayo Nozawa
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Correspondence to Dieter Söll or Osamu Nureki.

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Nozawa, K., O’Donoghue, P., Gundllapalli, S. et al. Pyrrolysyl-tRNA synthetase–tRNAPyl structure reveals the molecular basis of orthogonality. Nature 457, 1163–1167 (2009). https://doi.org/10.1038/nature07611

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