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.

About the Authors:

Lisa N. Kinch

* E-mail: [email protected]

Affiliation: Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America

Melanie L. Yarbrough

Affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America

Kim Orth

Affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America

Nick V. Grishin

Affiliations Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America

Introduction

The molecular function of fic (filamentation induced by cAMP) domains has remained elusive until recently. The original description of fic derives from the E. coli fic-1 gene required for cAMP-induced filamentation [1], [2]. Bacterial effector proteins that serve to modulate the activity of host cells also include fic domains. For example, the type IV secretion system effector AnkX from L. pneumophila has an N-terminal fic domain required for disrupting host secretory vesicle transport [3]. A fic domain is also present in VopS, a type III secretion system effector from V. parahaemolyticus that causes eukaryotic cell cytotoxicity [4]. The fic domain of VopS was shown to covalently modify host Rho GTPases with AMP. This AMPylation reaction occurred on a conserved threonine residue located in the GTPase Switch I region responsible for binding downstream effectors. Mutation of a conserved histidine in VopS abrogated this function, suggesting an enzymatic role for the residue in AMPylation [5].

AMPylation represents a newly discovered posttranslational modification used to stably modify proteins with AMP. This signaling mechanism is predicted to be functionally similar to other posttranslation modifications such as phosphorylation, SUMOylation or acetylation, because the added moiety changes the activity of the modified protein. The covalent attachment of AMP by a phosphodiester bond is predicted to be reversible and is bulky enough to provide a docking site for a putative AMP binding domain. Although a fic domain-containing protein is known to catalyze the AMPylation reaction, other components involved in this signaling system are yet to be discovered.

The fic domain is classified together with a second family of sequences, doc (death on curing), in the protein families database PFAM [6], and the sequences have been linked previously [7]. The combined family contains a central motif (fido) conserved in most sequences (HPFx[D/E]GN[G/K]R), with the motif His contributing to fic AMPylation [5]. Doc belongs to a toxin-antitoxin module found on E.coli phage P1 that acts as a plasmid addiction system. In the absence of phd antitoxin, doc induces growth arrest of E.coli by targeting protein translation elongation [8], [9]. Mutation of the motif His renders doc nontoxic, suggesting that the motif represents a functional site common to both families [10].

Structural genomics efforts have provided coordinates for four different hypothetical proteins with fic domains (2f6s, 2g03, 3cuc, and 3eqx), revealing a common α-helical fold topology [11]–[14]. Two of the fic domains (2f6s and 2g03) are related to the others by a circularly permuted helix. A similar α-helical structure of a non-toxic doc mutant (H66Y) bound to phd antitoxin (3dd7) replaces the permuted fic α-helix with an α-helical segment of phd. Together with the functional motif, the common topology of fic and doc domains suggests their evolution from a common ancestor [10].

To help resolve fido classification, we explored evolutionary relationships using a combination of sequence-based and structure-based methods. Sequence evidence supports an evolutionary link between fic and doc, while structural evidence supports a homologous relationship between these two families and a domain from the Pseudomonas syringae type III effector protein avirulence protein B (AvrB), which is delivered into plant cells to illicit accelerated defense responses and hypersensitive cell death by targeting the host immune resistance protein RIN4. We propose to unite domains from these three families (Fic, Doc, and AvrB) into a single superfamily (fido) that likely perform similar signaling functions. We show that like the VopS fic domain, a Drosophila melanogaster fic homolog self-AMPylates.

Results and Discussion

Classification of fido structures suggests homology to AvrB

Comparison of four available fic domain-containing hypothetical protein structures reveals a common fold topology that contributes to the evolutionary core of the domain (Figure 1A). The fic domain core includes eight α-helices, arranged as a six-helix up and down bundle (a1 - a5 and a') decorated by two additional helices (a6 and a7) lying almost perpendicular to the bundle. A β-hairpin inserted between helix a2 and a3 is positioned near the fido motif (loop between a4 and a5). In one fic-containing structure from Shewanella oneidensis (Figure 1B), a peptide from the N-terminus of a neighboring chain forms a β-like interaction with the hairpin [14]. The fic core of this structure is decorated by additional helices and includes a C-terminal domain not found in the other structures. One of the core α-helices (a') is permuted when compared to two closely related fic domains from Helicobacter pylori (Figure 1C) and Neisseria meningitidis (not shown), which contribute the a' helix from the C-terminus. Both the Shewanella oneidensis fic and the Bacteroides thetaiotaomicron fic (Figure 1D) include permuted a' helices contributed from the N-terminus and similar decorating helices.

[Figure omitted. See PDF.]

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).

https://doi.org/10.1371/journal.pone.0005818.g001

A doc domain structure bound to phd antitoxin revealed a similar overall topology to the fic domain [10]. However, doc lacks a few elements of the conserved fic core, including the first and the permuted α-helices and the β-hairpin (Figure 1E). The phd antitoxin replaces the two missing core α-helices, and was suggested to complete the core through fold complementation. Given the similar overall fold and the common motif positioned between corresponding α-helices, doc was suggested to have evolved from fic [10].

The fido structures display a similar topology that is somewhat unique among existing folds. To determine the degree of structural similarity between these folds and to help resolve existing classification schemes, we chose to compare each structure to existing structures in the PDB. The top hits (ordered by Dali Z-score) for each fic domain-containing query correspond to representative fic structures (Table 1). Despite recognized similarity to doc, the next best hit in each case represents a type III effector protein structure AvrB from Pseudomonas syringae. The AvrB structure is also the top hit using doc as a query, followed by the fic domains. Thus the fic, doc and AvrB structures form a “closed structural group”, displaying closer similarity to each other than to any other known protein structure. This property of structural similarity has been used previously to delineate EDD and SHS2 domains [15], [16] and is suggestive of monophyly.

[Figure omitted. See PDF.]

Table 1. Structural Similarity Search**.

https://doi.org/10.1371/journal.pone.0005818.t001

While the apo AvrB structure represented a novel fold with no recognizable homologs upon its release [17], a subsequent publication of RIN4 peptide-bound and nucleotide-bound AvrB structures [18] failed to consider the presence of fic domain structures in the PDB. AvrB retains all elements of the fic domain core, including the α-helices and β-hairpin missing in doc (Figure 1F). An additional domain is inserted in the β-hairpin that extends it into a five-stranded sheet referred to as the upper lobe [17]. A structure of AvrB bound to Rin4 reveals a similar peptide-binding mode as found in one of the fic structures (Figure 1B). The Rin4 peptide forms a β-like interaction with the extended sheet. Although the AvrB sequence lacks a number of the conserved fido-motif residues, it retains the small Pro and Gly motif residues that allow AvrB to adopt a similar loop conformation as in fic and doc structures (Figure 1, black). A number of AvrB mutations have been shown to alter the binding of Rin4. Two mutations in the upper lobe β-sheet (T125A and Q164H) and one mutation in the active site loop (S268I) abolish Rin4 binding, while another mutation in the active site pocket (D297A) enhances binding 10-fold [19]. Interestingly, the catalytically inactive VopS mutation also enhances its binding to GTPase by a “substrate trap” [5].

Sequence-based support for fido classification

To help confirm the implied evolutionary link between fic and doc, we used sequences corresponding to each domain as queries in transitive searches with PSI-BLAST [20]. The representative fic domain sequence from the B. thetaiotaomicron structure identified a number of doc sequences (ie. gi|159046642, detected in iteration 4, E-value 0.002) and converged without identifying sequences from other families. Similarly, the representative doc domain structure sequence identified a number of fic sequences (ie. gi| 195116213, detected in iteration 3, E-value 2e−04). The PSI-BLAST hits encompass a significant portion of the fic core and identified the conserved fido sequence motif (HPFx[D/E]GN[G/K]R). Identified fic and doc domain-containing sequences are present in genomes from bacteria, archaea, and eukaryota, with the latter distributed among metazoa species (HypE in human) and a few fungi. The phylogenetic distribution suggests extensive horizontal gene transfer (Figure S1), which represents a common observance for virulence and other genes involved in fitness [21].

Despite the structure similarity between AvrB and fido, most sequence methods fail to identify connecting links. However, using AvrB as a sequence query with the sensitive profile-based homology detection method COMPASS [22], [23] identified an insignificant hit (E-value 5) to the fic domain structure (3cuc). COMPASS accurately aligns the sequences of the two structures C-terminal to the inserted AvrB upper lobe domain, including the second strand of the β-hairpin, the active site loop, and helices a3 - a5. Apparently, the presence of an inserted domain within the AvrB β-hairpin, combined with an alteration of several active site motif residues hinders sequence recognition. Submitting an AvrB sequence that lacks the inserted domain to a fold-recognition meta-server (http://meta.bioinfo.pl) identifies fic domain structures as top hits with 3D-Jury scores [24] ranging from 48.83 for 3cuc to 60.83 for 2qc0 (scores correspond to approximately 50–60 correctly predicted residues [25]). The individual servers that identified fic domains from the truncated AvrB sequence (BASIC [26], SamT02 [27], and FFAS03 [28]) contain significant homology detection components.

The multiple sequence alignment illustrated in Figure 2 highlights the conservation patterns of representative fido domains. Each family displays similar hydrophobicity patterns (yellow highlights) corresponding to the secondary structural elements of the core fold. Conserved polar residues (black highlights) can be mapped to the structures: an R follows the α-helix a2, the fido motif HisPFx[D/E]G forms a loop connecting the two central α-helices a3 and a4, and the fido motif N[G/K]R forms the N-terminus of α-helix a4. A number of conserved positions in fic display different conservations in AvrB. The fido [D/E] is a conserved R in AvrB, while the fido N is a conserved S. A conserved Y in the α-helix a5 is positioned near a D in AvrB. In multiple sequence alignments of distant homologs, a similar “conservation of conservations” often reflects common functional sites [29]. Accordingly, mutation of AvrB R266 (corresponding to fido D/E) and D297 cause a loss of function leading to an abrogation of hypersensitive response in host plant cells, while mutation of T125 and H217 located in the peptide-binding β-hairpin abrogate both Rin4 interaction and AvrB induced function [18], [19].

[Figure omitted. See PDF.]

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.

https://doi.org/10.1371/journal.pone.0005818.g002

Fido domain organization suggests roles in stress response and cholesterol metabolism

Similar to other signaling proteins, fic and doc are fused to a number of different domains that might function in AMPlyation pathways (Figure 3). The Haemophilus somnus virulence factor p76 (ibpA) includes an N-terminal haemagglutination activity domain (Haem), two fic domains, and a papain-like protease (peptidase C58) also found in YopT and AvrPphB avirulence proteins. Like VopS, YopT disrupts the actin cytoskeleton of the host cell by inhibiting Rho GTPase signaling. YopT achieves this inhibition by cleaving Rho GTPases after a post-translationally modified cysteine, which releases the GTPases from the membrane where they function [30]. Thus, the H. somnus virulence factor includes multiple domains that modify the same signaling pathway. This functional domain organization suggests that other fusions might give similar clues to activity or help identify additional signaling components of AMPylation. The ibpA Haem domain is also found at the N-terminus of a doc domain containing protein from Comamonas testosteroni, providing further support for the homology and functional similarity between fic and doc.

[Figure omitted. See PDF.]

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.

https://doi.org/10.1371/journal.pone.0005818.g003

A number of fusions exist between fic domains and helix-turn-helix (HTH) DNA-binding domains. These HTH domains may regulate expression of fic or other components of AMPylation signaling. The DNA-binding component of these HTH domains is reminiscent of the phd antitoxin N-terminal DNA-binding domain that serves to autorepresses the doc toxin-antitoxin operon [31]. A majority of the HTH domains belong to the arsenical resistance operon repressor subfamily, indicating a potential role of fic in stress responses. We identify several additional HTH folds among domains of unknown function fused to fic (duf977, duf1612, and duf2089 in Figure 3). HHpred and COMPASS searches identify HTH folds as top significant hits to these domains: duf977 and the C-terminus of duf1612 find “winged helix” DNA-binding domains such as the 1j5y N-terminus (COMPASS E-value 5.84e-18) and the 1z05 N-terminus (COMPASS E-value 1.89e-14), while duf2089 finds C-terminal effector domains of the bipartite response regulators such as 1je8 (COMPASS E-value 6.97e-04). Interestingly, the N-terminus of duf1612 corresponds to remote fic-like sequences, and a member of this duf, Rhizobium Y4CF, belongs to a replicon that allows bacteria to interact symbiotically with leguminous plants [32]. The C-terminal domain from the S. oneidensis fic structure (Figure 2B, light green) represents a winged helix-turn-helix that is not recognized by sequence.

Eukaryotic fic sequences such as HypE contain a predicted N-terminal transmembrane helix followed by Tpr repeats. One interesting eukaryotic sequence from Branchiostoma floridae includes several additional domains that might hint at components involved in the eukaryotic fic domain signaling process. In addition to two fic and two Tpr, the B. floridae protein domain organization includes two low density lipoprotein receptor class a (LDL), two immunoglobulin (Ig), a trypsin-like serine protease (Trypsin), and five repeated calcium-binding EGF-like domains (EGF). The presence of these domains indicates an extracellular or intra vesicular localization of the pathway in eukaryotes, and the LDL might suggest involvment of AMPylation in cholesterol metabolism or trafficking.

Functional implications of Fido classification

Conserved fido motif residues (HPFx[D/E]GN[G/K]R) and additional conserved residues external to the motif (R50, R107, and Y132 in H. pylori fic) form a potential active site (Figure 4A and B). The external R50 residue organizes the fic active site, forming a number of hydrogen bonds to the backbone of the fido motif loop and to the peptide-binding β-hairpin. A phosphate residue forms hydrogen bonds to the fido motif helix backbone and the E100 and N102 side chains. A large pocket surrounds the bound phosphate, with the fido motif R104 lining a smaller cleft on one side of the bound phosphate and a Y132/R107 pair lining a larger cleft on the opposite side (Figure 4B). The smaller R104 cleft could accommodate ATP substrate phosphates, while the larger Y/R cleft could bind the sugar and nucleotide rings. The essential fido motif H96 is within 4 Å of the bound phosphate (Figure 4A) and lines a portion of the larger cleft in between the phosphate and peptide-binding β-hairpin (Figure 4B). This placement should position the fido His near the bound peptide sidechain that gets modified in the AMPylation reaction (Thr in the VopS modified Rho GTPase).

[Figure omitted. See PDF.]

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.

https://doi.org/10.1371/journal.pone.0005818.g004

Although the AvrB sequence lacks several residues of the fido motif, a conserved Arg (R117 in P. syringae AvrB) that corresponds to fic R50 maintains a similar local structure in the AvrB loop (Figure 4C). The conservations of small fido motif residues that extend to this loop in AvrB help maintain the local structure. R117 also forms hydrogen bonds to the Rin4 peptide-binding β-sheet. The α-phosphate of ADP binds AvrB in a similar position as the fic phosphate, forming hydrogen bonds to the corresponding motif helix backbone. The ADP binds in a cleft formed by conserved AvrB residues and the peptide-binding β-sheet (Figure 4D). An Arg residue (R266) takes the place of the fido motif D/E and forms hydrogen bonds with the ADP β-phosphate, while the Tyr of a Tyr/Arg pair (Y65/R99) forms hydrogen bonds with one of the ribose oxygens and stacks with the nucleotide ring. The positioning of both the R266 and the Y65/R99 pair has migrated with respect to the corresponding fic residues (R104 and Y132/R107, respectively), resulting in an active site pocket that is flipped. Despite the altered orientations of the nucleotide binding pockets, the ADP α-phosphate is near the peptide-binding site. AvrB is missing the essential fido motif His. However, several conserved residues in the AvrB active site could perform a similar function: D297, T125, or H217. D297 is located near the ADP phosphate. Mutation of this residue results in a similar increase of peptide-binding affinity as mutation of the fic His [5], [19]. T125 and H217 reside in the peptide-binding β-sheet and are positioned near a Rin4 Thr bound in the active site. Mutation of either of these residues results in altered AvrB activity [18], [19].

Activity of Fido domain-containing proteins

A previous study showed that a secreted protein from the pathogen V. parahaemolyticus catalyzes the addition of adenosine 5′-monophosphate (AMP) to Rho GTPases in a process known as AMPylation [5]. This activity represents the first example of using AMP as a stable post-translational modification on a eukaryotic substrate. Recombinant VopS possesses AMPylation activity even in the absence of substrate, as it is labeled by 32P-α-ATP in an in vitro AMPylation assay (Figure 5A). This auto-AMPylation activity is dependent on the presence of a wild-type fic domain, as mutation of the conserved His348 residue of the fic motif to an alanine completely abrogates activity (Figure 5A). In addition, an in vitro AMPylation assay with recombinant CG9523, a fic domain-containing protein from Drosophila melanogaster, indicates that CG9523 possesses auto-AMPylation activity (Figure 5B). As expected, mutation of the conserved His375 to alanine leads to a loss in the auto-AMPylation activity (Figure 5B). Thus, we have extended our observations to show that both bacterial and eukaryotic fic domains mediate the novel activity of AMPylation. Future studies on this and other eukaryotic fic domain-containing proteins and the identification of AMPylated substrates will provide insight into this novel posttranslational modification mediated by the fido domain-containing family of proteins.

[Figure omitted. See PDF.]

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.

https://doi.org/10.1371/journal.pone.0005818.g005

Conclusion

Bacterial virulence effectors use various strategies to manipulate host cell responses, often by mimicking structures or functions of eukaryotic signaling machinery. A secreted V. parahaemolyticus effector VopS mediates a newly discovered posttranslational modification that stably attaches AMP to host proteins. This AMPylation activity depends on a VopS histidine residue, which belongs to a conserved sequence motif characteristic of its fic domain. We use sequence and structure based similarity detection methods to identify fic domain homologs in doc toxins and the bacterial effector AvrB. Structures of these homologs help define the active site surrounding the critical histidine and the peptide-binding mode of AMPylation targets. Finally, we demonstrate AMPylation activity of a eukaryotic fic domain, suggesting that the VopS effector has exploited a previously unknown posttranslational modification for molecular signaling.

Materials and Methods

Sequence similarity searches

To detect sequence homologs of fic and doc domains, we searched the non-redundant database (nrf; posted date Nov 9, 2008; 7,269,299 sequences, filtered for low complexity regions) with PSI-BLAST [20] (E-value cutoff 0.005, iterated until convergence) using query sequences corresponding to the fic domain core of the Bacteroides thetaiotaomicron structure 3cuc (gi|186973118, range 66 to 275) and the doc domain of the structure 3dd7 (gi| 203282431, range 1 to 126). Found homologs were grouped using linkage clustering (-lscut = 0.9), and representative sequences from each group were used as transitive queries for subsequent rounds of PSI-BLAST. To detect sequence homologs of AvrB, we searched the non-redundant database with PSI-BLAST [20] (E-value cutoff 0.02) using query sequences corresponding to the AvrB structure 1nh1 (gi| 47168369, range 1 to 320). Found homologs were grouped using linkage clustering (-lscut = 1), and representative sequences from each group were used as new queries for subsequent rounds of PSI-BLAST. The COMPASS server [22], [23] and the HHpred server [33] were used to obtain more sensitive profile-based sequence searches for various queries: AvrB (gi|149242490), duf977 (gi|169755007), duf1612 (gi|198282242, range 250–387), and duf2087 (gi|218961370); and the fold recognition meta-server 3D-jury [24] was used to identify links to a truncated AvrB query (gi|149242490, range 55–128 and range 212–321) lacking the inserted upper lobe domain. Domain organization was assessed using the conserved domain database CDD [34] with a cutoff of 0.003.

Multiple sequence alignments and phylogenetic analysis

To visualize the relationships between identified fido sequences, all against all pair-wise sequence similarities for collected sequences (fic, doc, and AvrB) were calculated using BLAST implementation (-pval 0.1) with the CLANS application [35]. Sequences were clustered in two-dimensional space with a P-value cutoff of (1E-05) until node movement became negligible (rounds). Resulting clusters were used to constructed multiple sequence alignments using the MAFFT program (version 6, L-ins-I strategy, default parameters with no homologs [36], [37]) with manual adjustments. The multiple alignments of each group were merged into a global alignment using structure superpositions, secondary structure predictions (JPRED), hydrophobicity patterns, and paired BLAST hit alignments as guides.

Structural similarity searches

DaliLite [38] was used to determine the closest structural neighbors in the PDB database. For each query, all pairwise comparisons were then ranked in order of descending Z-score to determine the closest structural neighbors.

Plasmid construction and site-directed mutagenesis

CG9523 was amplified off of a plasmid containing the coding region of CG9523 (kindly provided by Helmut Kramer) using VENT polymerase. The PCR product was restriction digested, ligated into pET28a, and transformed into E. coli (DH5a, Invitrogen). Positive clones were confirmed by DNA sequence analysis. Histidine to alanine mutants of VopS and CG9523 were generated using the Stratagene QuikChangeTM site-directed mutagenesis kit, according to the manufacturer's instructions. Mutants were confirmed by DNA sequence analysis.

Protein expression and purification

VopSΔ30 and VopSΔ30-H348A were expressed and purified as described previously [5]. Protein expression constructs pET28a-CG9523 and pET28a-CG9523-H375A were transformed into Rosetta (DE3) cells (Novagen). Single colonies were grown to an OD600 of 0.6–0.8 and induced with 0.4 mM IPTG for 8–12 hours at room temperature. Cells were harvested and lysed using an Emulsiflex C-5 cell homogenizer (Avastin). His-tagged proteins (CG9523 and its mutant) were purified using Ni2+ affinity purification (Qiagen) and were aliquoted at stored at −80°C in 20 mM Tris, pH 7.5, 5 mM NaCl, 1 mM DTT, and 10% glycerol.

In vitro AMPylation assay

For the AMPylation assays using purified recombinant protein, 5 µM of VopSΔ30 or VopSΔ30-H348A or 2.5 µM of His-tagged CG9523 or His-tagged CG9523-H375A was incubated for 20 minutes at 30°C in 20 mM HEPES buffer containing 20 µM ATP, 2 mM MgCl2, and 32P-α-ATP (2 µCi) (Perkin Elmer). The AMPylation reaction was stopped by the addition of SDS sample buffer. Samples were boiled for 5 minutes, separated by SDS-PAGE, and visualized by autoradiography.

Supporting Information

[Figure omitted. See PDF.]

Figure S1.

Tree illustrates extensive horizontal gene transfer in bacteria/archaea. The multiple sequence alignment of one clans group was limited to sequence ranges corresponding to the fic topology of 3cuc and purged for redundancy less than 95% identity. The resulting alignment was used to construct phylogenetic trees using the MOLPHY package [39]. JTT distances [40] were calculated using protml (-jfD options), initial neighbor-joining [41] tree topologies were built using Njdist, and maximum likelihood trees were built using local rearrangement search of initial tree topologies protlm (−R option). The tree topology reliability was assessed with estimated log-likelihood resampling (RELL) of MOLPHY.

https://doi.org/10.1371/journal.pone.0005818.s001

(0.93 MB TIF)

Acknowledgments

We thank Helmut Kramer for kindly providing the CG9523 plasmid.

Author Contributions

Conceived and designed the experiments: LNK KO NG. Performed the experiments: MLY. Analyzed the data: LNK. Wrote the paper: LNK MLY. Performed computational experiments: LNK.

Citation: Kinch LN, Yarbrough ML, Orth K, Grishin NV (2009) Fido, a Novel AMPylation Domain Common to Fic, Doc, and AvrB. PLoS ONE4(6): e5818. https://doi.org/10.1371/journal.pone.0005818

References

1. Kawamukai M, Matsuda H, Fujii W, Nishida T, Izumoto Y, et al. (1988) Cloning of the fic-1 gene involved in cell filamentation induced by cyclic AMP and construction of a delta fic Escherichia coli strain. J Bacteriol 170: 3864–3869.M. KawamukaiH. MatsudaW. FujiiT. NishidaY. Izumoto1988Cloning of the fic-1 gene involved in cell filamentation induced by cyclic AMP and construction of a delta fic Escherichia coli strain.J Bacteriol17038643869

2. Komano T, Utsumi R, Kawamukai M (1991) Functional analysis of the fic gene involved in regulation of cell division. Res Microbiol 142: 269–277.T. KomanoR. UtsumiM. Kawamukai1991Functional analysis of the fic gene involved in regulation of cell division.Res Microbiol142269277

3. Pan X, Luhrmann A, Satoh A, Laskowski-Arce MA, Roy CR (2008) Ankyrin repeat proteins comprise a diverse family of bacterial type IV effectors. Science 320: 1651–1654.X. PanA. LuhrmannA. SatohMA Laskowski-ArceCR Roy2008Ankyrin repeat proteins comprise a diverse family of bacterial type IV effectors.Science32016511654

4. Ono T, Park KS, Ueta M, Iida T, Honda T (2006) Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system 1. Infect Immun 74: 1032–1042.T. OnoKS ParkM. UetaT. IidaT. Honda2006Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system 1.Infect Immun7410321042

5. Yarbrough ML, Li Y, Kinch LN, Grishin NV, Ball HL, et al. (2009) AMPylation of Rho GTPases by Vibrio VopS Disrupts Effector Binding and Downstream Signaling. Science 323: 269–272.ML YarbroughY. LiLN KinchNV GrishinHL Ball2009AMPylation of Rho GTPases by Vibrio VopS Disrupts Effector Binding and Downstream Signaling.Science323269272

6. Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, et al. (2008) The Pfam protein families database. Nucleic Acids Res 36: D281–288.RD FinnJ. TateJ. MistryPC CoggillSJ Sammut2008The Pfam protein families database.Nucleic Acids Res36D281288

7. Anantharaman V, Aravind L (2003) New connections in the prokaryotic toxin-antitoxin network: relationship with the eukaryotic nonsense-mediated RNA decay system. Genome Biol 4: R81.V. AnantharamanL. Aravind2003New connections in the prokaryotic toxin-antitoxin network: relationship with the eukaryotic nonsense-mediated RNA decay system.Genome Biol4R81

8. Lehnherr H, Maguin E, Jafri S, Yarmolinsky MB (1993) Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained. J Mol Biol 233: 414–428.H. LehnherrE. MaguinS. JafriMB Yarmolinsky1993Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained.J Mol Biol233414428

9. Liu M, Zhang Y, Inouye M, Woychik NA (2008) Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit. Proc Natl Acad Sci U S A 105: 5885–5890.M. LiuY. ZhangM. InouyeNA Woychik2008Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit.Proc Natl Acad Sci U S A10558855890

10. Garcia-Pino A, Christensen-Dalsgaard M, Wyns L, Yarmolinsky M, Magnuson RD, et al. (2008) Doc of Prophage P1 Is Inhibited by Its Antitoxin Partner Phd through Fold Complementation. J Biol Chem 283: 30821–30827.A. Garcia-PinoM. Christensen-DalsgaardL. WynsM. YarmolinskyRD Magnuson2008Doc of Prophage P1 Is Inhibited by Its Antitoxin Partner Phd through Fold Complementation.J Biol Chem2833082130827

11. (JCSG) JCfSG (to be published) Crystal structure of Protein of Unknown Function with a Fic Domain (NP_811426.1) from Bacteroides thetaiotaomicron VPI-5482 at 2.71 A resolution(JCSG) JCfSGto be publishedCrystal structure of Protein of Unknown Function with a Fic Domain (NP_811426.1) from Bacteroides thetaiotaomicron VPI-5482 at 2.71 A resolution

12. Cuff ME, Bigelow L, Bargassa M, Joachimiak A, Genomics MCfS (to be published) Structure of a putative cell filamentation protein from Neisseria meningitidisME CuffL. BigelowM. BargassaA. JoachimiakGenomics MCfSto be publishedStructure of a putative cell filamentation protein from Neisseria meningitidis

13. Cuff ME, Xu X, Zheng H, Edwards A, Savchenko A, et al. (to be published) Structure of cell filamentation protein (fic) from Helicobacter pyloriME CuffX. XuH. ZhengA. EdwardsA. Savchenkoto be publishedStructure of cell filamentation protein (fic) from Helicobacter pylori

14. Das D, Krishna SS, McMullan D, Miller MD, Xu Q, et al. (2009) Crystal structure of the Fic (Filamentation induced by cAMP) family protein SO4266 (gi|24375750) from Shewanella oneidensis MR-1 at 1.6 A resolution. Proteins 75: 264–271.D. DasSS KrishnaD. McMullanMD MillerQ. Xu2009Crystal structure of the Fic (Filamentation induced by cAMP) family protein SO4266 (gi|24375750) from Shewanella oneidensis MR-1 at 1.6 A resolution.Proteins75264271

15. Anantharaman V, Aravind L (2004) The SHS2 module is a common structural theme in functionally diverse protein groups, like Rpb7p, FtsA, GyrI, and MTH1598/TM1083 superfamilies. Proteins 56: 795–807.V. AnantharamanL. Aravind2004The SHS2 module is a common structural theme in functionally diverse protein groups, like Rpb7p, FtsA, GyrI, and MTH1598/TM1083 superfamilies.Proteins56795807

16. Kinch LN, Cheek S, Grishin NV (2005) EDD, a novel phosphotransferase domain common to mannose transporter EIIA, dihydroxyacetone kinase, and DegV. Protein Sci 14: 360–367.LN KinchS. CheekNV Grishin2005EDD, a novel phosphotransferase domain common to mannose transporter EIIA, dihydroxyacetone kinase, and DegV.Protein Sci14360367

17. Lee CC, Wood MD, Ng K, Andersen CB, Liu Y, et al. (2004) Crystal structure of the type III effector AvrB from Pseudomonas syringae. Structure 12: 487–494.CC LeeMD WoodK. NgCB AndersenY. Liu2004Crystal structure of the type III effector AvrB from Pseudomonas syringae.Structure12487494

18. Desveaux D, Singer AU, Wu AJ, McNulty BC, Musselwhite L, et al. (2007) Type III effector activation via nucleotide binding, phosphorylation, and host target interaction. PLoS Pathog 3: e48.D. DesveauxAU SingerAJ WuBC McNultyL. Musselwhite2007Type III effector activation via nucleotide binding, phosphorylation, and host target interaction.PLoS Pathog3e48

19. Ong LE, Innes RW (2006) AvrB mutants lose both virulence and avirulence activities on soybean and Arabidopsis. Mol Microbiol 60: 951–962.LE OngRW Innes2006AvrB mutants lose both virulence and avirulence activities on soybean and Arabidopsis.Mol Microbiol60951962

20. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.SF AltschulTL MaddenAA SchafferJ. ZhangZ. Zhang1997Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res2533893402

21. Dobrindt U, Hochhut B, Hentschel U, Hacker J (2004) Genomic islands in pathogenic and environmental microorganisms. Nat Rev Microbiol 2: 414–424.U. DobrindtB. HochhutU. HentschelJ. Hacker2004Genomic islands in pathogenic and environmental microorganisms.Nat Rev Microbiol2414424

22. Sadreyev R, Grishin N (2003) COMPASS: a tool for comparison of multiple protein alignments with assessment of statistical significance. J Mol Biol 326: 317–336.R. SadreyevN. Grishin2003COMPASS: a tool for comparison of multiple protein alignments with assessment of statistical significance.J Mol Biol326317336

23. Sadreyev RI, Tang M, Kim BH, Grishin NV (2007) COMPASS server for remote homology inference. Nucleic Acids Res 35: W653–658.RI SadreyevM. TangBH KimNV Grishin2007COMPASS server for remote homology inference.Nucleic Acids Res35W653658

24. Ginalski K, Elofsson A, Fischer D, Rychlewski L (2003) 3D-Jury: a simple approach to improve protein structure predictions. Bioinformatics 19: 1015–1018.K. GinalskiA. ElofssonD. FischerL. Rychlewski20033D-Jury: a simple approach to improve protein structure predictions.Bioinformatics1910151018

25. Kajan L, Rychlewski L (2007) Evaluation of 3D-Jury on CASP7 models. BMC Bioinformatics 8: 304.L. KajanL. Rychlewski2007Evaluation of 3D-Jury on CASP7 models.BMC Bioinformatics8304

26. Ginalski K, von Grotthuss M, Grishin NV, Rychlewski L (2004) Detecting distant homology with Meta-BASIC. Nucleic Acids Res 32: W576–581.K. GinalskiM. von GrotthussNV GrishinL. Rychlewski2004Detecting distant homology with Meta-BASIC.Nucleic Acids Res32W576581

27. Karplus K, Karchin R, Draper J, Casper J, Mandel-Gutfreund Y, et al. (2003) Combining local-structure, fold-recognition, and new fold methods for protein structure prediction. Proteins 53: Suppl 6491–496.K. KarplusR. KarchinJ. DraperJ. CasperY. Mandel-Gutfreund2003Combining local-structure, fold-recognition, and new fold methods for protein structure prediction.Proteins53Suppl 6491496

28. Jaroszewski L, Rychlewski L, Li Z, Li W, Godzik A (2005) FFAS03: a server for profile–profile sequence alignments. Nucleic Acids Res 33: W284–288.L. JaroszewskiL. RychlewskiZ. LiW. LiA. Godzik2005FFAS03: a server for profile–profile sequence alignments.Nucleic Acids Res33W284288

29. Mirny LA, Shakhnovich EI (1999) Universally conserved positions in protein folds: reading evolutionary signals about stability, folding kinetics and function. J Mol Biol 291: 177–196.LA MirnyEI Shakhnovich1999Universally conserved positions in protein folds: reading evolutionary signals about stability, folding kinetics and function.J Mol Biol291177196

30. Fueller F, Schmidt G (2008) The polybasic region of Rho GTPases defines the cleavage by Yersinia enterocolitica outer protein T (YopT). Protein Sci 17: 1456–1462.F. FuellerG. Schmidt2008The polybasic region of Rho GTPases defines the cleavage by Yersinia enterocolitica outer protein T (YopT).Protein Sci1714561462

31. Magnuson R, Lehnherr H, Mukhopadhyay G, Yarmolinsky MB (1996) Autoregulation of the plasmid addiction operon of bacteriophage P1. J Biol Chem 271: 18705–18710.R. MagnusonH. LehnherrG. MukhopadhyayMB Yarmolinsky1996Autoregulation of the plasmid addiction operon of bacteriophage P1.J Biol Chem2711870518710

32. Freiberg C, Fellay R, Bairoch A, Broughton WJ, Rosenthal A, et al. (1997) Molecular basis of symbiosis between Rhizobium and legumes. Nature 387: 394–401.C. FreibergR. FellayA. BairochWJ BroughtonA. Rosenthal1997Molecular basis of symbiosis between Rhizobium and legumes.Nature387394401

33. Soding J, Biegert A, Lupas AN (2005) The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res 33: W244–248.J. SodingA. BiegertAN Lupas2005The HHpred interactive server for protein homology detection and structure prediction.Nucleic Acids Res33W244248

34. Marchler-Bauer A, Anderson JB, Derbyshire MK, DeWeese-Scott C, Gonzales NR, et al. (2007) CDD: a conserved domain database for interactive domain family analysis. Nucleic Acids Res 35: D237–240.A. Marchler-BauerJB AndersonMK DerbyshireC. DeWeese-ScottNR Gonzales2007CDD: a conserved domain database for interactive domain family analysis.Nucleic Acids Res35D237240

35. Frickey T, Lupas A (2004) CLANS: a Java application for visualizing protein families based on pairwise similarity. Bioinformatics 20: 3702–3704.T. FrickeyA. Lupas2004CLANS: a Java application for visualizing protein families based on pairwise similarity.Bioinformatics2037023704

36. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30: 3059–3066.K. KatohK. MisawaK. KumaT. Miyata2002MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform.Nucleic Acids Res3030593066

37. Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9: 286–298.K. KatohH. Toh2008Recent developments in the MAFFT multiple sequence alignment program.Brief Bioinform9286298

38. Holm L, Park J (2000) DaliLite workbench for protein structure comparison. Bioinformatics 16: 566–567.L. HolmJ. Park2000DaliLite workbench for protein structure comparison.Bioinformatics16566567