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

Abstract

Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion with non-canonical amino acids, yet understanding of its structure and activity is incomplete. 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. Using standard microbiological culture equipment, we then established a new method for laboratory evolution – a non-continuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. We finally 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 non-canonical amino acid.

Linker and N-Terminal Domain Engineering of Pyrrolysyl-tRNA Synthetase for Substrate Range Shifting and Activity Enhancement

“…The T loop and the variable loop of tRNA Pyl have hydrophilic interactions with the PylRS-NTD. The tRNA Pyl is oppositely embraced by the NTD and the CTD of PylRS, which is in connection with the postulated flexible loops (Suzuki et al, 2017; Figure 1A). This compatible binding mode dynamically controls the interaction between PylRS and tRNA Pyl .…”

“…Moreover, the truncation of PylRS NTD significantly precludes the binding specificity of PylRS by making it incapable to charge tRNA Pyl (Jiang and Krzycki, 2012). The mutations on NTD of the evolved chimeric PylRS (chPylRS) have played a crucial role in tuning the binding of tRNA Pyl (Suzuki et al, 2017). The previous study of class II AARS seryl-tRNA synthetase (SerRS) showed that the R76 and the R94 of the NTD of MbSerRS were curial for tRNA recognition, and the alanine mutations of both had abolished the amber suppression efficiency (Jaric et al, 2009).…”

Synthesis at the interface of virology and genetic code expansion

“…[59] A variation of the same approach, where the phage-assisted directed evolution was performed in a discontinuous manner, also led to improved mutants. [60]…”

A high-throughput platform for feedback-controlled directed evolution

“…Phage persisted in all of the chPylRS-IP lagoons, and clonal phage acquired novel mutations (E301A or P5L). Only half of the chPylRS lagoons resulted in persistent phage propagation, but each acquired different mutations at the same N-terminal proline residue (P5L or P5T), within the conserved essential N-terminal domain of PylRS 30,31.…”