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 proteins. Furthermore, we show that the activity of Fic proteins promotes persister formation and that the ATPase activity of GyrB is blocked by Fic-mediated adenylylation of GyrB. As the structural consequences of ATP hydrolysis in GyrB remained elusive, I revealed in research article IV all nucleotide bound states along the ATP hydrolysis pathway by thorough X-ray crystallographic analysis. Upon ATP hydrolysis, an obligatory rigid-body domain motion of the transducer relative to the ATPase domain occurs in the immediate post-hydrolysis state (ADP⋅Pi).
In research article V, we dissect the regulation mechanism of the class III Fic protein NmFic that contains the αinh helix at the C-terminus. NmFic is in a monomer-tetramer equilibrium and structurally unable to bind a target in the tetramer form. Dissociation of the tetramer relieves the inhibition of this Fic protein and allows adenylylation of GyrB. Interestingly, NmFic is also auto-adenylylated. Auto-adenylylation of NmFic leads to conformational changes in the αinh helix and the neighboring α1 helix, with the Fic core being unchanged. We show that the presence of the strictly conserved tyrosine Y183 which is auto-adenylylated is crucial for both Fic protein activities. Thus, we propose that class III Fic proteins are tightly regulated by oligomerization and auto-adenylylation via a double-lock mechanism.
The inhibition of bacterial topoisomerases by Fic proteins of class I and III remains to be investigated at a structural level. I have shown that adenylylation of GyrB expulses the ATP-lid loop from its original position and I developed a new strategy to trap the Michaelis-Menten complex of a Fic protein and its cognate target by using covalent cross-linking of the Fic protein with the target via a functionalized ATP substrate.
Taken together, these results provide the first level of understanding of the regulation of FIC domain proteins and Fic-mediated inhibition of bacterial topoisomerases.