Data from: 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.