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, 13 (12), 1261-1266

Crystal Structures Reveal an Elusive Functional Domain of pyrrolysyl-tRNA Synthetase

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Crystal Structures Reveal an Elusive Functional Domain of pyrrolysyl-tRNA Synthetase

Tateki Suzuki et al. Nat Chem Biol.

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.

Conflict of interest statement

Competing financial interests

The authors declare competing financial interests: details accompany the online version of the paper.

Competing Financial Interests

The authors intend to file a patent application on the PANCE system.

Figures

Figure 1
Figure 1. Crystal structures of the wild-type and PANCE-evolved PylRS variants bound to tRNAPyl
(a) Superposition of the MmPylRS NTD•tRNAPyl complex (cyan; this study) onto the D. hafniense PylRS CTD•tRNAPyl complex (grey; PDB ID: 2ZNI). The M. mazei and D. hafniense tRNAPyl species have the same cloverleaf secondary structure but differ in 32% of their bases. The phosphorus atoms of 70% of the tRNAs (nt 1–7, 26–44, 49–72 comprising the acceptor stem, T-stem/loop and anticodon stem/loop of tRNAPyl) were used for the superposition (rmsd=4.04 Å). Zn2+ is represented as an orange sphere. The PylRS CTD is composed of catalytic (yellow) and tRNA binding (blue) domains. The Pyl recognition loop is indicated in pink. The possible path of the linker connecting NTD to CTD is shown by a black dashed line; the linker varies between 63–157 amino acids, depending on the source of PylRS. (b) Close-up view of the interface between MmPylRS NTD and tRNA Pyl. (c) Close-up view of the interface between PANCE-evolved PylRS variant 32A NTD and tRNA Pyl. Mutations evolved in the chPylRS NTD were transplanted into MmPylRS NTD for structure determination. PANCE-evolved residues are represented as spheres while nucleotides interacting with the NTD of each PylRS variant are given as sticks. Polar interactions between PylRS residues and tRNA nucleotides are illustrated by a black dashed line. Interaction distances are given in Å.
Figure 2
Figure 2. Structural basis for the PylRS specificity to tRNAPyl
(a) Superposition of the tRNAAsp (PDB ID: 2TRA) and tRNAPhe (PDB ID: 1EHZ) structures onto the tRNAPyl of the MmPylRS NTD•tRNAPyl complex was achieved by overlaying the phosphorus atoms of the acceptor stem, anticodon stem, and T–stem/loop (40 atoms for tRNAAsp, rmsd=3.04, and 41 for tRNAPhe, rmsd=3.21). MmPylRS NTD is shown as a surface model. (b) Close-up view of the variable loops of tRNAAsp and tRNAPyl (A44-U47and C45-G47, respectively). (c) Close-up view of the variable loops of tRNAPhe and tRNAPyl (A44-C48 and C45-G47, respectively). The steric clash between the MmPylRS NTD and the canonical tRNA is represented by a black dashed circle. Posttranscriptional modifications are omitted for clarity. (d) The cloverleaf structures of M. mazei tRNAPyl, S. cerevisiae tRNAAsp, and S. cerevisiae tRNAPhe show the different sizes of the variable loops.
Figure 3
Figure 3. PANCE method application and overview
(a) The PANCE method begins by first growing the host strain of E. coli until A600 = 0.3–0.5 in a large volume, before storing the cells at 4°C. An aliquot of 50 mL is then transferred to a smaller flask, supplemented with BocK and the inducing agent arabinose (Ara) for mutagenesis plasmid MP6, and is transfected with the selection phage (SP). This culture is incubated at 37°C for 8–12 hr to facilitate phage growth, which is confirmed by determination of the phage titer. Following phage growth, an aliquot of infected cells is used to transfect a subsequent flask containing host E. coli. This process is continued until the desired phenotype is evolved, for as many transfers as required. (b) PANCE improved BocK incorporation by chPylRS. Three independent lines of SP containing chPylRS were serially passaged across a host E. coli strain containing gIII. The number of UAG codons in gIII was steadily increased during serial passage in the presence of BocK. After 18–21 transfers, each lineage was able to survive with three UAG codons in gIII (P29am/E84am/Y183am) and grown in this environment for several more transfers to allow advantageous mutations to fix within the population. The lineages were named 32A, 24B, and 25C; the number denotes the number of transfers. For more details, see Online Methods.

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