Fido, a novel AMPylation domain common to fic, doc, and AvrB

Abstract

Background: The 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 findings: We 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/significance: The 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.

Figure 1. Structural similarities of fido domain-containing families.

Structural models of fic homologs define a core fic domain secondary structure topology labeled from N-terminus to C-terminus (A). Diverse fic domain-containing structures are illustrated from a Shewanella oneidensis [2qc0] (B), Helicobacter pylori [2f6s] (C), and Bacteroides thetaiotaomicron [3cuc] (D). The common core α-helices are colored in rainbow from N-terminus (blue) to C-terminus (red). A permuted helix is colored pink (contributed from the C-terminus) or slate (contributed from the N-terminus). Extended elements that decorate the core are colored white, a helix-turn-helix domain is colored light green, and a β-hairpin that binds peptide ligand (magenta) is colored gray. Bound ligands are represented as ball-and-stick and conserved sequence motifs marking the active sites are black. The N-terminus and the C-terminus of each structure is labeled. Similar structures retain most or all of the fic domain core: a doc structure [3dd7] bound to phd antitoxin (magenta) (E) and an AvrB structure bound to Rin4 peptide (magenta) [2nud] also binds ADP (ball-and-stick) [2nun, superimposed] (F).

Figure 2. Multiple sequence alignment of fido structures with representative sequences.

Fic, doc, and AvrB sequences are colored (blue, green, and magenta, respectively) and labeled with PDB ID (underlined), locus tag, or protein name. Positions corresponding to structurally conserved secondary structural elements are marked above the alignment (E for β-strand and H for α-helix). The number of the first residue position is indicated before each sequence, while omitted residues are in brackets. Uncharged residues at mainly hydrophobic positions are highlighted in yellow, and conserved small residues are highlighted in light gray. Conserved fic polar residues are highlighted black, along with the conservations that extended to doc or AvrB sequences. Moderate changes at conserved fic positions and conserved polar positions in AvrB that may represent active site migrations are highlighted in dark gray.

Figure 3. Fido Domains.

The domain organization of representative doc (green rectangle), fic (blue rectangle), and AvrB (magenta rounded rectangle) sequences is depicted. Abbreviations: RhsA, rhs repeat family protein; Hint, hedgehog/intein domain; Haem, haemagglutination activity domain; LRR, leucine rich repeat; HTH, regulatory helix-turn-helix; Tmh, transmembrane helix; Tpr, tetratrico peptide repeat; AAA, ATPase containing von Willebrand factor type A domain; LDL, Low-density lipoprotein receptor domain class A; Ig, Immunoglobulin-like; Trypsin, trypsin-like serine protease; EGF, calcium binding epidermal growth factor-like domain; Ribonuc Red sm, ribonucleotide reductase small subunit; DEXDc, DEAD-like helicase.

Figure 4. AvrB and Fic Active Site.

A zoom of the active site of fic (from 2f6s) colored light green in a ribbon diagram (A) and in a surface representation (B) and the active site of AvrB (from 2nun) colored slate in a ribbon diagram (C) and in a surface representation (D). Ribbon diagrams depict conserved residues as sticks that are labeled according to the H. pylori fic sequence or the P. syringae AvrB sequence and colored according to atom: Carbon (black), Oxygen (red), Nitrogen (blue). Gray dashed lines represent hydrogen bonds between active site residues and ligands, which are depicted as ball-and-stick and colored according to atom: Phosphorous (orange), Carbon (salmon), Oxygen (red), Nitrogen (blue). An α-phosphate of the AvrB ADP binds a similar position as the fic phosphate. Similar peptide binding β-hairpins (colored gray) reside near the active site. Residues labeled in white contribute to the active site pocket with their surface rendering not visible in the depicted orientation.

Figure 5. Auto-AMPylation of Fic domain-containing proteins.

Recombinant VopSΔ30 (WT) or VopSΔ30-H348A (H/A) from V. parahaemolyticus (A) and wild-type or mutant His-tagged CG9523 from D. melanogaster (B) were incubated with ³²P-α-ATP in an in vitro AMPylation assay. Samples were separated by SDS-PAGE and analyzed by autoradiography. The assay shows that wild-type, but not mutant Fic domain-containing proteins, possess auto-AMPylation activity.