Generating Efficient Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetases for Structurally Diverse Non-Canonical Amino Acids

Abstract

Genetic code expansion (GCE) technologies commonly use the pyrrolysyl-tRNA synthetase (PylRS)/tRNA Pyl pairs from Methanosarcina mazei (Mm) and Methanosarcina barkeri (Mb) for site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Recently a homologous PylRS/tRNA Pyl pair from Candidatus Methanomethylophilus alvus Mx1201 (Ma) was developed that, lacking the N-terminal tRNA-recognition domain of most PylRSs, overcomes insolubility, instability, and proteolysis issues seen with Mb/Mm PylRSs. An open question is how to alter Ma PylRS specificity to encode specific ncAAs with high efficiency.Prior work focused on "transplanting" ncAA substrate specificity by reconstructing the same active site mutations found in functional Mm/Mb PylRSs in Ma PylRS. Here, we found that this strategy produced low-efficiency Ma PylRSs for encoding three structurally diverse ncAAs: acridonyl-alanine (Acd), 3-nitro-tyrosine, and m-methyl-tetrazinyl-phenylalanine (Tet3.0-Me). On the other hand, efficient Ma PylRS variants were generated by a conventional life/death selection process from a large library of active site mutants: for Acd encoding, one variant was highly functional in HEK293T cells at just 10 μM Acd; for nitroY encoding, two variants also encoded 3-chloro, 3-bromo-, and 3-iodo-tyrosine at high efficiency; and for Tet-3.0-Me, all variants were more functional at lower ncAA concentrations. All Ma PylRS variants identified through selection had at least two different active site residues when compared with their Mb PylRS counterparts. We conclude that Ma and Mm/Mb PylRSs are sufficiently different that "active site transplantation" yields suboptimal Ma GCE systems. This work establishes a paradigm for expanding the utility of the promising Ma PylRS/tRNA Pyl GCE platform.

Structures of Methanomethylophilus alvus Pyrrolysine tRNA-Synthetases Support the Need for De Novo Selections When Altering the Substrate Specificity

“…The second molecule (chain B) had clear density only for ATP/AMPPNP in the Acd-RS1 structures and AMP (that we surmise arose from the hydrolysis of ATP during crystal growth) for the Acd-AST (Figure 4). No evidence was found for Acd binding in the second chain (chain B) for any structure even though Acd was present under saturating conditions (5 mM vs K D ∼ 0.3 mM) . Trapping of the dimer in this asymmetric state likely arose as a consequence of crystallization as two symmetrical protein molecules were not compatible with crystal packing (Figure S5).…”

“…No evidence was found for Acd binding in the second chain (chain B) for any structure even though Acd was present under saturating conditions (5 mM vs K D ∼ 0.3 mM). 20 Trapping of the dimer in this asymmetric state likely arose as a consequence of crystallization as two symmetrical protein molecules were not compatible with crystal packing (Figure S5).…”

“…In an accompanying paper, we describe a genetic code expansion (GCE) system derived from the Methanomethylophilus alvus Candidatus (Ma) pyrrolysine (Pyl) aminoacyl tRNA synthetase (PylRS)/tRNA Pyl pair for the efficient encoding of the fluorescent amino acid acridonyl-alanine (Acd, Figure A) at amber stop codons in E. coli and human cells.…”

“…Differences in Acd-RS1 are colored in red. (F) In-cell fluorescence of cells expressing sfGFP-150TAG in the presence and absence of Acd using Mb Acd-RS 41, the Ma Acd-AST, and Ma Acd RS1 derived from life/death selections (data shown is from our accompanying article). Data shown is the average of triplicates, and error bars represent the standard deviation.…”

“…To explore the limits of the AST strategy, we evaluated the efficiency of Ma PylRS ASTs harboring three or more mutations relative to wild-type for incorporating three aromatic ncAAs: acridonyl-alanine (Acd), 3-nitro-tyrosine, and mmethyl-tetrazinyl-phenylalanine (Tet3.0-Me). 20 In these cases, the AST strategy yielded Ma PylRS variants that were able to encode the targeted ncAAs but were inefficient compared to the Mb PylRSs from which they were derived, whereas much more efficient Ma PylRS variants were generated from de novo selections. Specifically, for Acd, sequence analysis of the top selected Ma Acd-PylRS (referred to as Acd-RS1) revealed three residue differences compared to the Ma Acd-AST (Figure 1E).…”