Fido, a Novel AMPylation Domain Common to Fic, Doc, and AvrB

Abstract

BackgroundThe Vibrio parahaemolyticus type III secreted effector VopS contains a fic domain that covalently modifies Rho GTPase threonine with AMP to inhibit downstream signaling events in host cells. The VopS fic domain includes a conserved sequence motif (HPFx[D/E]GN[G/K]R) that contributes to AMPylation. Fic domains are found in a variety of species, including bacteria, a few archaea, and metazoan eukaryotes.Methodology/Principal FindingsWe show that the AMPylation activity extends to a eukaryotic fic domain in Drosophila melanogaster CG9523, and use sequence and structure based computational methods to identify related domains in doc toxins and the type III effector AvrB. The conserved sequence motif that contributes to AMPylation unites fic with doc. Although AvrB lacks this motif, its structure reveals a similar topology to the fic and doc folds. AvrB binds to a peptide fragment of its host virulence target in a similar manner as fic binds peptide substrate. AvrB also orients a phosphate group from a bound ADP ligand near the peptide-binding site and in a similar position as a bound fic phosphate.Conclusions/SignificanceThe demonstrated eukaryotic fic domain AMPylation activity suggests that the VopS effector has exploited a novel host posttranslational modification. Fic domain-related structures give insight to the AMPylation active site and to the VopS fic domain interaction with its host GTPase target. These results suggest that fic, doc, and AvrB stem from a common ancestor that has evolved to AMPylate protein substrates.

Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and 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.…”

“…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.…”

A New Chemical Handle for Protein AMPylation at the Host–Pathogen Interface

“…Key bacterial AMPylators characterized to date include VopS, a cytotoxin of Vibrio parahaemolyticus, that AMPylates Rho family GTPases at a conserved threonine residue; [9] IbpA secreted by Histophilus somni, that AMPylates the same target protein but modifies a tyrosine residue instead of a threonine; [10] and finally DrrA (also known as SidM), a Legionella pneumophila effector that AMPylates Rab1 GTPases also at a tyrosine. [11] Both VopS and IbpA mediate AMPylation using a so-called Fido catalytic motif (canonical sequence: [12] and target the host cell cytoskeleton, whilst DrrA utilizes an adenylyl transferase domain (canonical sequence: Gx11DxD) [11] and targets host cell protein trafficking. In 2011, the first de-AMPylating enzyme, or 'protein AMPylase', was discovered in L. pneumophila, confirming the importance and the dynamic nature of posttranslational AMPylation.…”

A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism

“…Also, the Fic domains can be found either as single domain proteins or as individual domains in multidomain proteins. A typical Fic domain consists of a structural core of eight ␣-helices described as a six-helix up-anddown bundle (␣1-␣5 and ␣Ј1) with helices ␣6-␣7 lying roughly perpendicular to the bundle (18). The highly conserved and essential histidine of the Fic motif is the catalytic residue believed to deprotonate the attacking hydroxyl group of the target protein (19,21,27).…”

“…The highly conserved and essential histidine of the Fic motif is the catalytic residue believed to deprotonate the attacking hydroxyl group of the target protein (19,21,27). Fic proteins also include the flap, either a ␤-hairpin or a loop, bridging helices ␣2-␣3, proposed to mediate substrate binding (18,19,23). Recently, it was shown that adenylylation activity of some Fic domains is controlled by a conserved mechanism of ATP binding site obstruction involving an intrinsic inhibitory ␣-helix (␣ inh ) containing a conserved (S/T)XXXE inh (G/N) inhibitory motif (21).…”