Pyrrolysyl-tRNA synthetase-tRNA(Pyl) structure reveals the molecular basis of orthogonality

Abstract

Pyrrolysine (Pyl), the 22nd natural amino acid, is genetically encoded by UAG and inserted into proteins by the unique suppressor tRNA(Pyl) (ref. 1). The Methanosarcinaceae produce Pyl and express Pyl-containing methyltransferases that allow growth on methylamines. Homologous methyltransferases and the Pyl biosynthetic and coding machinery are also found in two bacterial species. Pyl coding is maintained by pyrrolysyl-tRNA synthetase (PylRS), which catalyses the formation of Pyl-tRNA(Pyl) (refs 4, 5). Pyl is not a recent addition to the genetic code. PylRS was already present in the last universal common ancestor; it then persisted in organisms that utilize methylamines as energy sources. Recent protein engineering efforts added non-canonical amino acids to the genetic code. 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 tRNA(Pyl) function as an orthogonal pair in vivo. Here we show that Desulfitobacterium hafniense PylRS-tRNA(Pyl) is an orthogonal pair in vitro and in vivo, and present the crystal structure of this orthogonal pair. The ancient emergence of PylRS-tRNA(Pyl) 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.

Figure 1

Overall structures. (a) DhPylRS subunit A, shown as a ribbon model, consisting of the tRNA binding domain 1 (1.68, blue), the catalytic domain (69.96 and 128.266, beige), the bulge domain (97.127, yellow), and the C-terminal tail (267.288, light green), with the motif 2 loop (160–170) colored red and the Pyl recognition loop (212–218) colored purple. (b) The dimeric DhPylRS:tRNA^Pyl complex structure, shown as a ribbon model. The asymmetric unit contains one PylRS dimer and two tRNA^Pyl molecules; PylRS-A (colored as in panel a), PylS-B (gray), tRNA-I (blue), and tRNA-II (pink). (c) Binding of tRNA^Pyl (ribbon representation) to the surface model of DhPylRS, structural domains are colored as in (a).

Figure 2

Class I (a) and class II (b) tRNA synthetase:tRNA complexes are structurally aligned. Only a single monomer of the catalytic core domains are displayed, color coded according to structural similarity. Viewed from the major groove side of the acceptor stem, a phosphate backbone outline of the tRNAs is shown (tan), and tRNA^Pyl is shown in purple. In space filling representation, a glutamyl-adenylate (a) and a pyrrolysyl-adenylate (b) highlight the class I and class II active site pockets, respectively. The small substrates are partially obscured by the protein backbone due to the need to show both aaRS families in the same orientation relative to the tRNA.

Figure 3

DhPylRS:tRNA^Pyl interface. (a) View showing the recognition of the CCA terminus by the motif-2 loop of DhPylRS. (b) View showing the recognition of the tRNA^Pyl acceptor helix by DhPylRS. (c,d) Views showing the recognition of the tRNA^Pyl minimal core by the core-binding surface of DhPylRS.

Figure 4

Comparison of PylRS structures: (a) DhPylRS:tRNA^Pyl complex, (b) DhPylRS apo, (c) MmPylRS:Pyl-AMP complex (PDB code 2zim), (d) MmPylRS:ATP, Cyc complex (2q7g), (e) MmPylRS:ATP analog complex (2q7e), (f) MmPylRS apo (2e3c). The structures are colored according to B-factor (indicating more [red] or less [blue] structurally dynamic regions), and four regions that show conformational changes in the different structures are labeled in panel a. Only one subunit of the dimer is shown for clarity.