Engineered reactivity of a bacterial E1-like enzyme enables ATP-driven modification of protein C termini

In biological systems, ATP provides an energetic driving force for peptide bond formation, but protein chemists lack tools that emulate this strategy. Inspired by the eukaryotic ubiquitination cascade, we developed an ATP-driven platform for C-terminal activation and peptide ligation based on E. coli MccB, a bacterial ancestor of ubiquitin-activating (E1) enzymes that natively catalyzes C-terminal phosphoramidate bond formation. We show that McCB can act on non-native substrates to generate an O-AMPylated electrophile that can react with exogenous nucleophiles to form diverse C-terminal functional groups, including thioesters, a versatile class of biological intermediates that have been exploited for protein semisynthesis. To direct this activity towards specific proteins of interest, we developed the Thioesterification C-terminal Handle (TeCH)-tag, a sequence that enables high-yield, ATP-driven protein bioconjugation via a thioester intermediate. By mining the natural diversity of the MccB family, we developed two additional MccB/TeCH-tag pairs that are mutually orthogonal to each other and to the E. coli system, facilitating the synthesis of more complex bioconjugates. Our method mimics the chemical logic of peptide bond synthesis that is widespread in biology for high-yield in vitro manipulation of protein structure with molecular precision.

Methods
This deposition includes 1) plasmid maps for expression constructs used in the relevant manuscript; and 2) raw data from an Agilent LC-TOF MS instrument in the *.d format. Raw LC-TOF MS data were processed in Agilent MassHunter BioConfirm v10.0 (intact protein data) or Agilent MassHunter Qualitative Analysis 10.0 (peptide data). The data deposited here has not been processed. For experiments described in our manuscript, intact protein spectra were deconvoluted using the Maximum Entropy algorithm. Peptide data were analyzed by extracting spectra or generating extracted ion chromatograms.