Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria

Abstract

Tyrosine phosphorylation is a common protein posttranslational modification, which plays a critical role in signal transduction and the regulation of many cellular processes. Using a pro-peptide 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 the 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 crystal structure of the synthetase reveals a reconfigured substrate binding site formed by non-conservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site whose corresponding kinase is unknown and determined 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.

Incorporation of Phosphorylated Tyrosine into Proteins: In Vitro Translation and Study of Phosphorylated IκB-α and Its Interaction with NF-κB

“…20,21,24 Several laboratories have used enzymatic methods in conjunction with native chemical ligation to produce phosphorylated proteins in vitro , 39 and there have also been modified phosphorylated proteins produced by other combinations of enzymatic and chemical methods. 40 Recently, the incorporation of phosphotyrosine into a protein in cellulo has been reported, 41 and the incorporation of phosphoserine has also been reported. 42 It seems reasonable to anticipate that expression in cells containing the modified ribosomes of the type described here might increase the yield of phosphorylated proteins elaborated in cells.…”

Playing with the Molecules of Life

“…11–16 The orthogonality of this pair must be such that the aaRS does not recognize endogenous tRNAs (84 in Escherichia coli ,) or amino acids, and only aminoacylates its cognate tRNA; additionally, the tRNA must not act as a substrate for any endogenous aaRSs. 3, 17 The final key component is the ncAA itself, which can often be directly supplemented to the growth media, taken up as a dipeptide precursor through a transporter, 18 or biosynthesized by the host. 19 An elegant example of the synthesis of all these components was the genetic encoding of p -aminophenylalanine in engineered bacteria that contained the orthogonal aaRS/tRNA pair, an amber codon inserted into the myoglobin sequence, and a gene cluster from S. venezuelae that enables the bacterial biosynthesis of p- aminophenylalanine to afford a 21 amino acid synthetic organism.…”

“…A number of post-translational modifications have been genetically encoded, including phosphoserine, phosphothreonine, phosphotyrosine, sulfotyrosine, nitrotyrosine, and numerous epigenetic lysine modifications. 18, 26, 60, 71, 74–77, 240, 263–266 Stable mimicks of posttranslational modifications, such as phosphonotyrosine and carboxymethyl-phenylalanine have also been genetically encoded. 72, 75, 267 Incorporation of this latter phosphotyrosine mimetic into the arginine methyltransferase PRMT1 facilitated analysis of how phosphorylation alters the binding of this protein to its substrates.…”

“…268 Phosphonotyrosine was recently genetically incorporated into proteins by exploiting the polyspecificity of the previously evolved carboxymethyl-phenylalanine aaRS, and a dipeptidyl transporter to transport this negatively-charged amino acid into cells. 269 Subsequent incorporation of phosphonotyrosine into human Abl1 at specific residues allowed determination of the binding affinities of the various phosphorylated forms of the protein to its substrates. Additionally, 3-nitrotyrosine has been genetically encoded, and used to gain new insights into post-translational modifications that modulate inflammatory responses and mimic disease-state processes.…”

The ABCs of PTMs

“…This means that OTS-installed PTMs can be recognized and eliminated by enzymes in mammalian systems and other organisms. For this reason, some ncAA sidechains consisting of nonhydrolyzable analog versions of PTMs have been genetically encoded 22,23 . Future strategies to improve modified protein synthesis in native contexts may rely on genome engineering to create new orthogonal synthesis compartments that sequester modified proteins from catabolic enzymes, or new cell lines that exhibit less hydrolytic activity (e.g.…”