Investigation of phosphorylating, ADP-ribosylating and glycosylating bacterial toxins specificities via reactive protein-proteome profiling "RP3

This proposal describes the future development of Reactive Protein - Proteome Profiling “ RP3 ”, a concept we pioneered for over the last years. The RP3 method allows for the absolute substrate profiling of bacterial toxins against cellular target proteins.

The RP3-concept employs covalently linked binary nucleotide co-substrate - toxin complexes as warheads, thus resulting in covalent ternary complexes upon enzymatic reaction with protein substrates. The method can be seen as a macromolecular version of Activity-based Proteome Profiling (ABPP). During infection, pathogenic bacteria tweak their eukaryotic hosts by translocating toxins.

Toxins often display catalytic transferase activity against host cell proteins, targeting key functions like cell signaling, vesicular transport and cytoskeleton dynamics.

Toxins make use of reactive metabolites, like nucleotides, as co-substrates - which offers us a chance to chemically modify the system. No chemical tools exist for the proteomic evaluation of the absolute substrate profile of a given toxin. Here, the RP3-method fills the gap.

The RP3-approach also allows for the creation of covalently tethered ternary complexes of protein / toxin combinations with low native affinity, thus for the first time enabling structural investigations of otherwise inaccessible toxin-substrate complexes.

In this project, we will expand the RP3 concept into phosphorylating- ADP-ribosylating- and glycosylating toxins from pathogenetic bacteria.