Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria

A propeptide strategy increases uptake of phosphotyrosine and a nonhydrolyzable analog to facilitate their incorporation into proteins by recombinant methods, aided by a specific aminoacyl-tRNA synthetase with a reconfigured active site. Tyrosine phosphorylation is a common protein post-translational modification that plays a critical role in signal transduction and the regulation of many cellular processes. Using a propeptide strategy to increase cellular uptake of O -phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl- L -phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase–tRNA pair that allows site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray structure of the synthetase reveals a reconfigured substrate-binding site, formed by nonconservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site and determining the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.