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

Abstract

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 αinh 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 αinh 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.

Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification

“…Probably, the segment flanking the Tyr residue would engage in β-strand interaction with the flap, resulting in correct registration of the modifiable tyrosyl side-chain within the active site. This binding mode would be analogous to the one seen in the complex structures of IbpA Fic2 /cdc42 (6) and of noncognate peptide/Fic proteins (9). Because the flap is partially buried at the center of the tetramer, the structural model also explains why the tetramer would be incompetent for automodification.…”

“…Automodification has been observed for most Fic proteins described to date (6,(8)(9)(10)(11)(12)(13)(14)(15). For NmFic, the sites of autoadenylylation have been mapped to Y183 and Y188 of the α inh by mass spectrometry (MS) (8) ( Fig.…”

“…Proteins were expressed and purified as described (8,25). Toxicity tests and adenylylation assays were performed according to the protocols given by Harms et al (5) and Goepfert et al (9).…”

“…However, the biological functions, as well as the molecular mechanism, of the vast majority of Fic proteins have remained elusive. Intriguingly, in vitro automodification has been demonstrated for most Fic proteins that have been described so far (6,(8)(9)(10)(11)(12)(13)(14)(15), but its physiological relevance has remained unclear.…”

“…These three possibilities lead to a classification of Fic proteins into classes I, II, and III, respectively (8). Mutation of the inhibitory Glu to Gly relieves autoinhibition, thus boosting both target and autoadenylylation (8,9). However, the identity of the intrinsic or extrinsic factors that in vivo expulse the inhibitory Glu of α inh , and thereby relieve autoinhibition, has not been investigated.…”