Structure - function studies on FIC - mediated AMPylation and deAMPylation by class I Fic proteins

Proteins containing a conserved FIC (filamentation induced by cyclic AMP) domain can be found in all domains of life, where they modify the function of target proteins via post-translational modifications such as AMPylation describing the transfer of AMP onto the threonine, tyrosine, or serine side chain of their respective targets.
First studies on AMPylation activity by two bacterial proteins VopS from Vibrio parahaemolyticus and IbpA from Histophilus somni revealed inhibition of RhoGTPases causing disruption of the actin cytoskeleton leading to cell death. Since then, the AMPylation activity of several Fic proteins containing a highly conserved FIC domain has been described. Fic proteins are controlled by the presence of an inhibition motif and depending on its location the enzymes can be separated into three classes. Proteins such as human FICD or NmFic from Neisseria meningitidis have this inhibition motif either on the N-terminus or the C-terminus of the toxin itself and represent class II and class III Fic proteins, respectively. FICD mediates AMPylation of the Hsp70 chaperone BiP in the endoplasmatic reticulum (ER), which leads to BiP’s inactivation when the level of unfolded proteins is low. Recently it was shown that FICD deAMPylates BiP and removes the modification, which recruits the target back into chaperone cycle when the load of unfolded proteins is high. Studies on the class III Fic protein EfFic revealed deAMPylation activity of the protein and that Fic proteins can act as bifunctional enzymes. A conserved glutamate in the inhibition motif of both proteins plays a significant role in regulation of the reactions. Class I Fic proteins such as VbhT from Bartonella schoenbuchensis are inhibited by a small protein antitoxin consisting of the inhibition motif. Another example for class I Fic proteins are the effector proteins of the alpha-proteobacterial genus Bartonella, which have a growing number of species and are studied as model for evolution of bacterial pathogenesis. Bartonella effector proteins (Beps) contain a diverse ensemble of FIC domains, which have evolved in parallel in three Bartonella lineages from a single ancestral toxin-antitoxin module.
In research article I we use X-ray crystallography, structural modelling, and phylogenetic analysis to gain more insight into the variety of Beps, which includes nine crystal structures and 99 non-redundant sequences. Minor structural changes of the core FIC domain indicate functional and regulatory variability of Beps.
In research article II we show that a recent developed nucleotide quantification assay is a sensitive method to obtain real-time enzymatic progress curves. This assay was chosen to characterize the AMPylation and deAMPylation reaction mediated by the FIC domain of the class I toxin VbhT (VbhT(FIC)) from Bartonella schoenbuchensis, which is regulated by its cognate antitoxin VbhA. Autoradiography assays previously revealed that VbhT(FIC) AMPylates the DNA gyrase subunit B (GyrB) leading to its inactivation and abolishing cell growth, which is inhibited when the antitoxin was present. We show that the VbhT/VbhA toxin-antitoxin complex acts as a bifunctional enzyme causing AMPylation and deAMPylation of a 43 kilodalton (kDa) subunit of GyrB (GyrB43). Mutation of the glutamate in the inhibition motif, which is known to have strong modifying effects, reveals enhanced AMPylation and deAMPylation activity, indicating a different role of the glutamate in the two reactions compared to class II FICD and class III EfFic.