Tightly regulated FIC domain proteins modulate bacterial topoisomerases by adenylylation

Proteins containing FIC (filamentation induced by cAMP) domains are conserved through evolution and found ubiquitously in all domains of life and viruses. Fic proteins catalyze adenylylation, or AMPylation, the transfer of an adenosine-5’-monophosphate moiety (AMP) from an ATP substrate onto a target protein. The adenylylation activity of two bacterial proteins, VopS from Vibrio parahaemolyticus and IbpA from Histophilus somni was discovered recently. VopS and IbpA are translocated into eukaryotic cells and adenylylate the small GTPases RhoA, Rac1 and Cdc42 in the switch I region, resulting in the inhibition of downstream effector binding and ultimately collapse of the actin cytoskeleton and cell death. Due to their cytotoxic activity in eukaryotic cells but also in bacteria, Fic proteins need to be tightly regulated. To contribute to the understanding of the regulation mechanism of Fic proteins and inhibition of their targets, I applied biochemical, biophysical and mainly X-ray crystallographic analysis. This work was achieved in close collaboration with microbiologists. In research article I, we show that adenylylation competent Fic proteins containing the HxFx[D/E]GNGRxxR motif are inhibited by a conserved α-helix (αinh) that contains a [S/T]xxxE[G/N] inhibition motif. The αinh helix can be found on a separate protein that forms a tight complex with the Fic protein, or at the N-terminus or C-terminus compared to the Fic active site. These three possibilities lead to the classification of Fic proteins into class I, II and III, respectively. The strictly conserved glutamate of this motif competes with the binding of the γ-phosphate of the ATP substrate of Fic proteins. In research article II, we structurally demonstrate that this inhibitory mechanism applies independent of the position of the αinh helix relative to the Fic active site motif. In the research article III we identify GyrB and ParE, the B-subunits of the bacterial topoisomerases DNA gyrase and topoIV, as new bacterial targets for class I Fic ...