On plasmids, TA modules act as addiction systems, aiding plasmid maintenance in the bacterial population by post-segregational killing (6), filling in a function related to apoptosis and programmed cell death in eukaryotes (7). Related effects have been observed for chromosome-located TA systems as some of them have been shown to diminish large scale genome reductions in the absence of selection (8). In the presence of the plasmid, both toxin and antitoxin are expressed, leading to a steady state equilibrium where the antitoxin counteracts the effect of the toxin. In its free state, the antitoxin, usually a modular protein that contains a functional intrinsically disordered region (9–11), is under constant proteolytic attack. The toxin-antitoxin complex acts as an autorepressor for the TA operon, ensuring that only small amounts of the proteins are present in the cell. Upon plasmid loss, the antitoxin is degraded by a specific intracellular protease, releasing the toxin. Without the possibility of replenishing the antitoxin population, the toxin action becomes irreversible, resulting in cell death.

The phd/doc operon encodes a TA module aiding the maintenance of the plasmid-prophage P1 in Escherichia coli (12). Doc is an inhibitor of translation elongation through its association with the 30 S ribosomal subunit in a way similar to the antibiotic hygromycin (13). The action of Doc is suppressed by the antitoxin Phd, which consists of two domains. Its C-terminal domain (residues 52–73) harbors the interaction site with Doc and on its own prevents Doc-mediated growth arrest (14, 15). The N-terminal region (residues 1–51) of Phd is a dimerization/DNA-binding domain that binds to the operator site of the phd/doc operon. Phd forms a heterotrimeric complex with Doc (16). Operator binding and repression of the phd/doc operon by Phd are enhanced by the presence of Doc in a cooperative manner (17–19).

CD Spectropolarimetry—Far UV-CD spectra were recorded on a J-715 spectropolarimeter (Jasco). Scans were taken using a 0.1-cm cuvette. The temperature of the cuvette was monitored using a probe, and a water bath was used for maintaining the temperature of the sample constant. The measurements were performed at 298 K, in 50 mm Tris (pH 7.5), 150 mm sodium chloride. Spectra of Doc^H66Y and Phd^52–73Se were taken using a protein concentration of 60 μm. For the spectrum of the complex, Doc^H66Y and Phd^52–73Se were mixed in equimolar ratio to a final concentration of 60 μm of the complex and preincubated for 5 min before taking the spectrum. The mean residue ellipticities ([θ], degrees cm² mol^-1) were obtained from the raw data (θ, ellipticity) after correcting for the buffer solution, according to [θ] = θ·Mw/(n·c·l), where Mw is the molecular weight, c is the mass concentration, l is the optical path length, and n is the number of amino acid residues.

Multiple sequence alignment of Doc family members reveals a single highly conserved motif, HXFX(D/E)(A/G)N(K/G)R. It is located in the loop α3-α4 (residues 66–74) (Fig. 1), and its conformation can be described as two consecutive NEST motifs. These are short structural elements that are often used as anion-binding sites (26) (supplemental Fig. S2a). This sequence motif forms the core of a single patch of conserved surface residues (Fig. 2A) that further includes residues from the loop α1-α2 as well as His-13. The latter plays a structural role for establishing the correct conformation of the conserved sequence motif. In addition, a number of mutations known to eliminate Doc toxicity but that retain co-repression activity (H66Y, H66R, and D70N) map within this motif (Fig. 2B) (19). This is a clear indication for a functional role of this region, likely an interaction site.

Interactions with Phd—The C-terminal domain of Phd on its own is sufficient to protect against Doc (14, 15). Phd^52–73Se binds into a groove of Doc^H66Y of which helix α4 forms the base and that is flanked by helix α1 on one side and the loop α4-α5 on the other side (Fig. 1A). This binding site is adjacent to the hotspot of conserved residues on the surface of Doc but by itself is not highly conserved (Fig. 2A). The binding groove has an approximate volume of 2050 ų and contains a large number of positively charged side chains (Arg-2, Arg-19, Lys-73, Arg-74, Arg-85, and Arg-86) around a hydrophobic center. As such, it forms a positively charged patch on the otherwise overall negatively charged surface of Doc (Fig. 2C).

Far UV CD experiments show that Phd^52–73Se is intrinsically unstructured in its isolated state but gains an appreciable amount of α-helix upon binding to Doc (Fig. 3). In the crystal structure, Phd^52–73Se adopts an α-helical conformation when bound to Doc and shields about 820 Ų of the Doc surface from the solvent. The interactions between Phd^52–73Se and Doc^H66Y are mediated entirely by side chain atoms (Fig. 4A). A kink in the α-helix divides Phd^52–73Se into a hydrophobic N-terminal segment (residues 54–62) and a predominantly negatively charged C-terminal segment (residues 64–73) (Fig. 4B). In its folded state, bound to Doc, the N-terminal segment of Phd^52–73Se is distinctly amphipathic with its hydrophobic side interacting with Doc. The side chains Leu-59, Phe-60, and Leu-63 of Phd^52–73Se become completely buried on this hydrophobic surface, creating an extension of the hydrophobic core of Doc. Phe-56, which is more exposed, extends this set of interactions by packing against Leu-12 and Leu-81 of Doc (Fig. 4).

The C-terminal segment of Phd^52–73Se is highly hydrophilic and provides only a single hydrophobic residue (Leu-70 in Phd, Se-Met-70 in Phd^52–73Se) to the binding interface. This residue makes extensive contacts with a small hydrophobic cavity on the Doc surface (Fig. 4B). The Phd-Doc contact surface in this region shows a high degree of charge complementarity with several negatively charged side chains of Phd^52–73Se (Glu-55, Asp-61, and Asp-64) interacting favorably with positive residues in the Phd-binding groove of Doc^H66Y (Arg-19 and Arg-85). Most striking in this region of Phd^52–73Se are the interactions involving Asn-67. This residue is completely buried in the interface, its side chain protruding inside a small hydrophilic pocket where it makes complementary hydrogen bonds with the side chain of Asn-16 and Asn-78 of Doc (Fig. 4).

It should be noted here that although the C terminus of Phd^52–73Se is adjacent to the surface cluster of conserved residues, the conserved sequence motif of Doc is not part of the Phd-binding site. This indicates that Phd^52–73Se counteracts the toxic activity of Doc either by inducing a conformational change in Doc or by sterically preventing Doc to interact with the ribosome. Both mechanisms have been proposed earlier on for other TA modules (27, 28).

Doc Has an Incomplete Fic Fold—Structural similarity searches against the Structural Classification of Proteins-(SCOP) data base using the DALI server failed to reveal any protein with significant similarity to Doc. However, all Doc homologues possesses a conserved central motif (see above) of 9 residues that is shared with two other protein families: the bacterial cAMP-induced filamentation protein (Fic) and a domain of the eukaryotic Huntingtin Yeast Protein E (HYPE) protein (29). Sequence similarity between Doc and these other two protein families outside this 9-residue region is very weak with overall sequence identities below 15%.

A query of the Protein Data Bank with the conserved central motif of Doc resulted in the identification of two proteins that show a high structural similarity to Doc. These otherwise undescribed recent depositions from the Midwest Center for Structural Genomics are crystal structures of two Fic proteins: Fic_Hp from Helicobacter pylori (PDB entry 2F6S) and Fic_Nm from Neisseria meningitidis (PDB entry 2G03). Fic_Nm is the most closely related to Doc with an root mean square deviation of 2.3 Å for 105 matching Cα atoms (Z score 6.7) (Fig. 5A). A structure-based sequence alignment is given in Fig. 5B.

Fic in most respects resembles the architecture of Doc, including very similar conformations for the conserved central motif in loop α3-α4, suggesting a common evolutionary origin (supplemental Fig. S2). Also, His-13, the residue that in Doc anchors the conserved central motif, is conserved and makes equivalent interactions in all structures (supplemental Fig. S2). Nevertheless, Fic differs from Doc by the presence of an extra N-terminal α-helix, by an insertion in the loop α1-α2, and most importantly, by an additional C-terminal α-helix (Fig. 5A). The latter adopts a position and conformation in the Fic structure that is strikingly similar to the position and conformation of the Phd peptide in the Doc-peptide complex (Fig. 5A). Removing Phd^52–73Se from the complex unveils a large hydrophobic patch that extends toward the hydrophobic core. This is suggestive of an incomplete protein, and indeed, free Doc is poorly soluble and prone to aggregation and misfolding.

Based upon these observations, we propose that Doc evolved from a Fic-like ancestor of which the C-terminal α-helix was transferred to a DNA-binding domain, thereby generating the antitoxin Phd. Upon binding, the antitoxin donates an α-helix to form the complex thereby complementing the fold.

Doc Induces Growth Arrest but Not Cell Death—Induction of Doc leads to growth arrest within the doubling time of E. coli. However, the cells do not lyse and remain motile for several hours after induction, when examined under a light microscope. This indicates an intact cell membrane and a working proton motive force. We do not observe filamentation in cells in which Doc has been activated, although filamentation but not induction of the SOS pathway was reported earlier (12). The observed growth arrest is reversible as cells replated in the absence of IPTG are capable of colony formation for several hours after the start of IPTG induction. We further confirmed that Doc arrests bulk protein synthesis but not RNA or DNA synthesis (Fig. 6). Additionally, we observed that Doc inhibits protein synthesis in a cell-free expression system and that Phd prevents this inhibition (data not shown).

Doc Induces RelE-mediated Cleavage of Model mRNAs—A recent study showed that Doc expression in E. coli strain BL21(DE3) led to mRNA stabilization (13). By contrast, we observe destabilization of two different model mRNAs (lpp and dksA) after induction of doc in E. coli strain MG1655 (Fig. 7A and supplemental Fig. S3). Primer extension analysis after doc induction of lpp and dskA mRNAs reveals cleavage patterns very similar to that induced by RelE, especially just downstream of the start codons (Fig. 7B; supplemental Figs. S3 and S4). However, in contrast to RelE, Doc does not induce mRNA cleavages near the stop codons. Strikingly, non-translated versions of the lpp and dskA mRNAs (start codons changed to AAG) are not affected by induction of doc (Fig. 7A and supplemental Figs. S3 and S4). Thus, like RelE-induced cleavage, Doc-induced mRNA cleavage depends on translation. These results suggest that the cleavage is due to the activation of endogenous TA loci rather than a direct effect of Doc itself. Indeed, Doc-induced mRNA cleavage is not observed in a strain deficient in the five major E. coli TA modules (mazEF, chpBIK, relBE, yoeB/yefM, and dinJ/yafQ-MG1655Δ5) (Fig. 7B, lanes 7–8). Moreover, expression of Doc in a strain that lacks relBE (MG1655ΔrelBE) also fails to induce Doc-mediated mRNA cleavage (Fig. 7B, lanes 11–12), indicating that ectopic production of Doc activates endogenous RelE. A previous report (30) indicated that Doc induces MazF activity. However, we did not observe mRNA cleavage at ACA sites, the signature sequence of MazF-mediated mRNA cleavage (31). Neither do we observe any influence of deleting mazEF on the cleavage pattern. Therefore our results do not confirm that Doc activates MazF. RelE has previously been described to be activated during nutritional stress due to Lon-mediated degradation of RelB antitoxin. The Doc-induced cleavage sites depend on Lon (Fig. 6B, lanes 9 and 10). These results are consistent with the proposal that the Doc-mediated inhibition of translation leads to RelE activation via Lon-dependent decay of RelB. Thus, the mRNA decay seen after induction of doc appears to be an indirect consequence of Lon-dependent activation of RelE. Consistent with this hypothesis is the observation by Liu et al. (13) that mRNA is not destabilized by induction of doc in E. coli strain BL21(DE3). BL21(DE3) is optimized for protein production and lacks the Lon and OmpT proteases (32).

Doc has been shown to inhibit protein synthesis and to associate with the 70 S ribosome and with the 30 S ribosomal subunit (13). The mechanism of action of Fic is currently unknown. The strong structural similarities between both proteins and the presence of a highly conserved and functionally important region raise the possibility that Fic is also capable of halting translation on the ribosome or to modify ribosomal activity. Available data indicate that Fic has a role in cell division (33), most likely under the tight control of the cell division machinery. Mutations of Fic, such as G55R (34), may distort this control, leading to its accidental activation (12, 33). We suggest that Doc evolved from a mutant of a Fic-like ancestor defective in regulation of its activity. Likely, the C terminus of such an ancestor of Doc was transferred to a DNA-binding domain. This resulted in a novel regulatory system that by its ability to be used for conditional killing or growth arrest can function as a plasmid addiction module and as a TA stress operon. The existence of several open reading frames of doc-like genes fused to genes encoding DNA-binding domains among different bacterial genomes favors this hypothesis. Further studies are nevertheless required to substantiate this hypothesis.

During complex formation, Phd donates its C-terminal domain to complement the truncated Fic-like fold of Doc. Fold complementation has been described as one of the major mechanisms by which proteins can bind peptides in a β-strand conformation (35). As a result of the complex formation, the added β-strand complements for a missing secondary structure element in an otherwise incomplete fold. The completion of an Ig-like β-sandwich in the subunit-subunit and chaperone-subunit interactions in bacterial pili assembled by the chaperoneusher pathway and the addition of the hepatitis C virus NS4A cofactor peptide to the N-terminal β-sheet in the NS3 protease that complements a chymotrypsin-like fold are classical examples of this mechanism (36, 37). To our knowledge, this mechanism has not been observed before for α-helical peptide ligands binding to all-α-helical proteins. The amphipathic nature of the α-helix of Phd and the hydrophobic surface patch at the center of the Phd-binding site on Doc are likely remnants of the Fic-like ancestor of Doc, which had its hydrophobic core partially disrupted when it lost its C terminus.

Our in vivo experiments indicate that the ectopic overexpression of Doc can induce the mRNA interferase activity of RelE, a chromosomal TA toxin of E. coli. Activation of RelE, together with MazF, is triggered by stress conditions and is probably part of the general response of the cell to internal alarm signals. RelE activation may be an attempt of the cells to relieve the stress induced by Doc-mediated translation arrest. Indeed, RelE activity correlates with tmRNA activity (38). Thus, mRNA degradation on Doc-arrested ribosomes may mimic translation quality control systems that are invoked when termination of translation fails.