Conserved Inhibitory Mechanism and Competent ATP Binding Mode for Adenylyltransferases with Fic Fold

The ubiquitous FIC domain is evolutionarily conserved from bacteria to human and has been shown to catalyze AMP transfer onto protein side-chain hydroxyl groups. Recently, it was predicted that most catalytically competent Fic proteins are inhibited by the presence of an inhibitory helix α_inh that is provided by a cognate anti-toxin (class I), or is part of the N- or C-terminal part of the Fic protein itself (classes II and III). In vitro, inhibition is relieved by mutation of a conserved glutamate of α_inh to glycine. For the class III bacterial Fic protein NmFic from Neisseria meningitidis, the inhibitory mechanism has been elucidated. Here, we extend above study by including bacterial class I and II Fic proteins VbhT from Bartonella schoenbuchensis and SoFic from Shewanella oneidensis, respectively, and the respective E->G mutants. Comparative enzymatic and crystallographic analyses show that, in all three classes, the ATP substrate binds to the wild-type FIC domains, but with the α-phosphate in disparate and non-competent orientations. In the E->G mutants, however, the tri-phosphate moiety is found reorganized to the same tightly bound structure through a unique set of hydrogen bonds with Fic signature motif residues. The γ-phosphate adopts the location that is taken by the inhibitory glutamate in wild-type resulting in an α-phosphate orientation that can be attacked in-line by a target side-chain hydroxyl group. The latter is properly registered to the Fic active center by main-chain β-interactions with the β-hairpin flap. These data indicate that the active site motif and the exposed edge of the flap are both required to form an adenylylation-competent Fic protein

Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins

Fic proteins that are defined by the ubiquitous FIC (filamentation induced by cyclic AMP) domain are known to catalyse adenylylation (also called AMPylation); that is, the transfer of AMP onto a target protein. In mammalian cells, adenylylation of small GTPases through Fic proteins injected by pathogenic bacteria can cause collapse of the actin cytoskeleton and cell death. It is unknown how this potentially deleterious adenylylation activity is regulated in the widespread Fic proteins that are found in all domains of life and that are thought to have critical roles in intrinsic signalling processes. Here we show that FIC-domain-mediated adenylylation is controlled by a conserved mechanism of ATP-binding-site obstruction that involves an inhibitory ?-helix (?(inh)) with a conserved (S/T)XXXE(G/N) motif, and that in this mechanism the invariable glutamate competes with ATP ?-phosphate binding. Consistent with this, FIC-domain-mediated growth arrest of bacteria by the VbhT toxin of Bartonella schoenbuchensis is intermolecularly repressed by the VbhA antitoxin through tight binding of its ?(inh) to the FIC domain of VbhT, as shown by structure and function analysis. Furthermore, structural comparisons with other bacterial Fic proteins, such as Fic of Neisseria meningitidis and of Shewanella oneidensis, show that ?(inh) frequently constitutes an amino-terminal or carboxy-terminal extension to the FIC domain, respectively, partially obstructing the ATP binding site in an intramolecular manner. After mutation of the inhibitory motif in various Fic proteins, including the human homologue FICD (also known as HYPE), adenylylation activity is considerably boosted, consistent with the anticipated relief of inhibition. Structural homology modelling of all annotated Fic proteins indicates that inhibition by ?(inh) is universal and conserved through evolution, as the inhibitory motif is present in ?90% of all putatively adenylylation-active FIC domains, including examples for all domains of life and from viruses. Future studies should reveal how intrinsic or extrinsic factors modulate adenylylation activity by weakening the interaction of ?(inh) with the FIC active site