Pyrrolysyl-tRNA synthetase–tRNAPyl 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 tRNAPyl1. 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 catalyzes the formation of Pyl-tRNAPyl4,5. 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 demonstrate 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 for the evolution of unique structural features in both the protein and the tRNA. These structural elements manifest an intricate, specialized aaRS:tRNA interaction surface highly distinct from those observed in any other known aaRS:tRNA complex; it is this general property that underlies the molecular basis of orthogonality.

Identification of Amino Acids in the N-terminal Domain of Atypical Methanogenic-type Seryl-tRNA Synthetase Critical for tRNA Recognition

“…Most notably, class I and class II aaRSs (including pyrrolysyl-tRNA synthetase, see Ref. 9) approach tRNAs from the minor and major groove sides of the acceptor stem, respectively (10). Although the majority of determinants are in direct contact with cognate synthetases (8), the aminoacylation fidelity is controlled by kinetic differences more than by binding affinities (11).…”

PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine

“…The structure of D. hafniense PylSc is similar to that of M. mazei ⌬185PylS, although with an apparently tighter binding pocket for pyrrolysine (13,30). PylSc binds tRNA Pyl primarily through interactions with residues of the acceptor and D stem with no direct contact to residues of the T arm, anticodon arm, or variable loop (13).…”

“…The secondary structure of tRNA Pyl is unusual, having a small D loop and variable loop and an elongated anticodon stem, as well as lacking the base typically found between the acceptor and D stems (9). Nonetheless, tRNA Pyl assumes the expected L-shaped tertiary structure but with a more compact core relative to most tRNA species (12,13).…”

“…The deletion was necessary to eliminate the highly basic yet hydrophobic N-terminal portion of the protein, whose presence interfered with protein stability and crystallization (29). The structure of D. hafniense PylSc is similar to that of M. mazei ⌬185PylS, although with an apparently tighter binding pocket for pyrrolysine (13,30). PylSc binds tRNA Pyl primarily through interactions with residues of the acceptor and D stem with no direct contact to residues of the T arm, anticodon arm, or variable loop (13).…”

Reducing the genetic code induces massive rearrangement of the proteome

“…The PylRS enzyme was engineered to genetically encode >100 ncAAs (19). The Pyl encoding system has already been used to expand the genetic codes of Escherichia coli (20)(21)(22), mammalian cells, and animals (23).…”