Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase

Abstract

Highlights d A mutant pyrrolysyl-tRNA synthetase, PylRS(Y306A/Y384), acts on diverse amino acids d The PylRS mutant and tRNA Pyl incorporated 17 non-natural amino acids into proteins d Crystal structures of the PylRS mutant bound with 14 of the amino acids were solved d This information will facilitate the structure-based design of novel amino acids

Phosphine‐Activated Lysine Analogues for Fast Chemical Control of Protein Subcellular Localization and Protein SUMOylation

“…To better understand OABKRS′s efficient incorporation of MPABK , as well as the binding modes of PABK , MOABK , and MPABK to their respective synthetases, we conducted a docking study using AutoDock Vina (Supporting Information). Modeling revealed that MPABK ′s side chain is kinked such that the benzylic methyl group fits into a space at the top of the tunnel, and the para ‐azido substituent is accommodated facing outward in the pocket toward D373 (Figure S4 A and D), nearly identically to the azide in a published co‐crystal structure of meta ‐azido‐ N ϵ ‐Cbz‐Lys in OABKRS . PABK ′s azide group is positioned similarly.…”

“…This is one of the most versatile PylRS mutants for incorporating sterically demanding lysine derivatives; the Y349F mutation increases aminoacylation kinetics and yield, while Y271A exposes a deep hydrophobic pocket in the PylRS catalytic site that accommodates benzyl carbamates . This synthetase incorporated MOABK as well (Figure S3), but produced only modest protein yields with PABK (Figure S2 A) . A PylRS mutant with L274A, C313A, and Y349F mutations was significantly more efficient at accepting PABK as a substrate (Figure S2 B) and was termed PABKRS .…”

“…A PylRS mutant with L274A, C313A, and Y349F mutations was significantly more efficient at accepting PABK as a substrate (Figure S2 B) and was termed PABKRS . OABKRS prefers ortho ‐ and meta ‐substituted N ϵ ‐Cbz‐Lys derivatives over para ‐substituted substrates; PABK was no exception to this rule (Figure S2 A). To better understand OABKRS′s efficient incorporation of MPABK , as well as the binding modes of PABK , MOABK , and MPABK to their respective synthetases, we conducted a docking study using AutoDock Vina (Supporting Information).…”

“…As an added advantage, however, in addition to providing control over protein function via the Staudinger reduction, the azide‐containing UAAs reported here may be activated with other reagents (such as TCOs and Ru II complexes). Further, they may function as vibrational reporters for IR spectroscopy, may reveal transient or weak protein–protein interactions by photo‐crosslinking, and could serve as handles for bioconjugation via cycloaddition reactions . Thus, these next‐generation azido‐lysine derivatives serve as multifunctional, efficiently incorporated handles for activating and probing protein structure and function in mammalian cells.…”

Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids

“…Site-saturation mutagenesis (SSM) via NNK codons (N: A/T/C/G; K:G/T) was used to specifically randomize chosen positions of the aaRS [ 23 ]. Our strategy is further based on an analysis of the available high-resolution molecular structure of Mm PylRS, as well as earlier reports showing that mutagenesis of the crucial positions N346 and C348 ( Figure 2 ) is feasible to expand the substrate range of this enzyme [ 24 , 25 ]. We make a step further and include other residues in our mutagenesis schemes, importantly including second and higher shell residues, which are expected to be important for ncAA substrate binding and recognition, but not in direct contact.…”