Genetically Encoding Phosphotyrosine and Its Nonhydrolyzable Analog in Bacteria

Nat Chem Biol. 2017 Aug;13(8):845-849. doi: 10.1038/nchembio.2405. Epub 2017 Jun 12.

Abstract

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.

MeSH terms

  • Codon, Nonsense / genetics*
  • Crystallography, X-Ray
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Ligases / chemistry
  • Ligases / metabolism
  • Models, Molecular
  • Molecular Structure
  • Phosphorylation
  • Phosphotyrosine / analogs & derivatives*
  • Phosphotyrosine / genetics*
  • Phosphotyrosine / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism

Substances

  • Codon, Nonsense
  • Recombinant Proteins
  • Phosphotyrosine
  • Ligases

Associated data

  • PubChem-Substance/336297846
  • PubChem-Substance/336297847
  • PubChem-Substance/336297848
  • PubChem-Substance/336297849
  • PubChem-Substance/336297850
  • PubChem-Substance/336297851
  • PubChem-Substance/336297852