Parst Is a Widespread Toxin–Antitoxin Module That Targets Nucleotide Metabolism

ParST is a widespread toxin–antitoxin module that targets nucleotide metabolism

Frank J. Piscottaa, Philip D. Jeffreyb, and A. James Linka,b,c,1

aDepartment of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544; bDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544; and cDepartment of Chemistry, Princeton University, Princeton, NJ 08544

Edited by Marlene Belfort, University at Albany, Albany, NY, and approved December 4, 2018 (received for review August 27, 2018) Toxin–antitoxin (TA) systems interfere with essential cellular pro- I toxins are typified by an RNA-level TA interaction, where an cesses and are implicated in bacterial lifestyle adaptations such as antisense RNA binds toxin mRNA to inhibit its translation. Type persistence and the biofilm formation. Here, we present structural, II TA systems function via protein–protein interactions, where biochemical, and functional data on an uncharacterized TA system, binding of the antitoxin to the toxin inhibits its activity (10). Of the COG5654–COG5642 pair. Bioinformatic analysis showed that the two earliest known TA systems mentioned above, hok/sok is this TA pair is found in 2,942 of the 16,286 distinct bacterial species an example of a type I system, while ccdAB is type II (11, 12). in the RefSeq database. We solved a structure of the toxin bound Upon translation, type I toxins are small, hydrophobic peptides to a fragment of the antitoxin to 1.50 Å. This structure suggested that lead to cell lysis through disruption of the plasma mem- that the toxin is a mono-ADP-ribosyltransferase (mART). The toxin brane. Type II toxins, however, function through a variety of specifically modifies phosphoribosyl pyrophosphate synthetase mechanisms. A large group of these are endoribonucleases that (Prs), an essential enzyme in nucleotide biosynthesis conserved cleave RNA in either a ribosome-dependent or independent in all organisms. We propose renaming the toxin ParT for Prs manner. The well-studied HigB, RelE, and MazF toxins, among ADP-ribosylating toxin and ParS for the cognate antitoxin. ParT many others, all fall into this group (13–15). Beyond these, is a unique example of an intracellular protein mART in bacteria however, there is much variation. HipA phosphorylates glutamyl- and is the smallest known mART. This work demonstrates that TA tRNA synthetase (GltX), inactivating it, while CcdB engages in systems can induce bacteriostasis through interference with nucle- gyrase-mediated double-stranded DNA cleavage (16, 17). An- otide biosynthesis. other toxin, FicT, also interferes with DNA gyrase, but rather by inactivation through adenylation (18). More recently, DarT was toxin–antitoxin system | ADP-ribosylation | posttranslational modification found to ADP-ribosylate single-stranded DNA (19). The continued discovery of new toxins like DarT emphasizes the role of ur understanding of toxin–antitoxin (TA) systems has pro- TA systems in cell biology. Ogressed significantly since their identification nearly 35 y Type II TA systems are quite prevalent throughout bacterial ago. In the pioneering work, ccdAB was shown to enhance the genomes. In a comprehensive analysis by Makarova et al. (20) in stability of a mini-F plasmid in Escherichia coli by killing 2009, of the 750 reference genomes searched, putative type II daughter cells lacking the plasmid and also established that the TA systems were found in 631, with on average 10 loci in each hit ccdA genetic element could provide an antidote for the post- (6,797 total). The number of occurrences in a single genome can segregational killing (PSK) of ccdB (1). An investigation into the be much larger, however; the deadly pathogen Mycobacterium E. coli R1 plasmid revealed a similar system hok/sok that also tuberculosis has over 88 TA loci in its genome, while the soil improved plasmid stability, where again one element, sok, could bacterium and plant symbiont Sinorhizobium meliloti contains neutralize the PSK activity of the other, hok (2). Results such as an astounding 211 predicted type II loci (21, 22). Makarova these led to the hypothesis that the role of such genetic regions et al. proposed the existence of 19 new putative type II TA pairs, was to ensure the inheritance of plasmids during cell division. It was later discovered, however, that these systems resided not Significance only in plasmids but also on bacterial chromosomes and that, while not conferring replicon stability, they may play an impor- – tant role in regulating cell growth (3–5). Further investigations Toxin antitoxin (TA) systems are pairs of genes found throughout into bacterial TA systems have also implicated them in biofilm bacteria that function in DNA maintenance and bacterial sur- vival. The toxins in these systems function by inactivating growth and persister formation. The toxin gene mqsR was first critical cell processes like protein synthesis. This work describes shown to be up-regulated in E. coli biofilms and later confirmed a TA system found in 17% of all sequenced bacteria. A high- to promote biofilm formation through regulation of quorum resolution crystal structure and an array of biochemical tests sensing (6, 7). One of the most ubiquitous TA systems, hipBA, revealed that this toxin targets an essential enzyme in nucle- was the first to be implicated in persister formation; the well- otide biosynthesis, a unique way in which TA systems can halt characterized mutant hipA7 was shown to increase E. coli per- bacterial growth. sistence up to 1,000-fold, due to a weakened interaction with its

antitoxin (8). Various other toxins have also been implicated in Author contributions: F.J.P. and A.J.L. designed research; F.J.P., P.D.J., and A.J.L. per- biofilm and persister formation, including RelE, YafQ, and formed research; F.J.P., P.D.J., and A.J.L. analyzed data; and F.J.P. and A.J.L. wrote MazF (9). As the importance of biofilms and persisters in clinical the paper. settings grows, the study of TA systems provides an avenue for The authors declare no conflict of interest. better understanding the mechanisms driving these processes, as This article is a PNAS Direct Submission. well as a possible path to intervention. Published under the PNAS license. Although most downstream effects of these toxins are gener- Data deposition: The atomic coordinates and structure factors have been deposited in the ally similar (bacteriostasis or cell death), the methods by which Protein Data Bank, www.wwpdb.org (PDB ID codes 6D0I and 6D0H). these systems work are remarkably diverse. TA systems are 1To whom correspondence should be addressed. Email: [email protected]. broadly grouped into six classes, type I through VI, based on the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. nature of the TA interaction, although type I and II comprise 1073/pnas.1814633116/-/DCSupplemental. most known TA systems and have been extensively studied. Type Published online December 31, 2018.

826–834 | PNAS | January 15, 2019 | vol. 116 | no. 3 www.pnas.org/cgi/doi/10.1073/pnas.1814633116 Downloaded by guest on October 2, 2021 including 12 new toxins and antitoxins, again highlighting the cludes the structure of an RES domain protein. Solving this structure variety that exists within the type II class. To organize the rapidly allowed us to determine that the toxin is a mono-ADP-ribosyl- increasing amount of data available, the TA database was cre- transferase (mART). We further show, both in vivo and in vitro that ated in 2010 and is an invaluable resource in parsing what we the toxin ADP-ribosylates phosphoribosyl pyrophosphate synthetase know about these systems (23). (Prs), an essential enzyme involved in nucleotide biosynthesis. This Of the 19 predicted TA systems, one stood out to us in par- report expands the set of bacteriostatic mechanisms employed by ticular. The COG5654 (RES domain)-COG5642 TA family is TA systems to include protein-modifying ADP-ribosylation. one of five putative TA systems from this paper where both components were uncharacterized. We had encountered this Results pair in genome mining for natural products studied by our lab- Sphingobium sp. YBL2 Bears Multiple COG5654–COG5642 TA Systems. oratory and were intrigued by how poorly understood it was Our investigation into Sphingobium sp. YBL2 began when a considering how widespread it appeared to be; it was present in genome-mining algorithm developed in-house for discovering 250 of the 750 genomes analyzed by Makarova et al. Following new lasso peptide producers identified this organism as having its identification in 2009, we found only one study that examined two lasso peptide gene clusters of interest (24). It was immedi- the putative COG5654–COG5642 TA pair, a genetic study on ately evident that one of these gene clusters differed from the S. meliloti. This organism has 211 putative TA loci as mentioned expected architecture, containing within it genes encoding two above, with roughly 100 of these on the organism’s two mega- hypothetical proteins, yblI and yblJ (Fig. 1A). A BLAST of these plasmids. The authors demonstrated through a series of genetic sequences revealed them to be a COG5654–COG5642 putative deletions that only four of these were actively functioning as TA TA pair, first identified in a computational study in 2009 and systems, including a COG5654–COG5642 locus, which con- later confirmed to function as a TA pair in S. meliloti (20, 22). To firmed the hypothesis put forth in Makarova’s work (22). our knowledge, this is the last published work on this TA family. Here, we report a detailed physiological, structural, and bio- COG5654 toxins contain a roughly 150-aa RES domain, chemical study of the COG5654–COG5642 TA system, starting named for a highly conserved Arg-Glu-Ser motif. In addition, the with a computational approach that demonstrates its prevalence domain also features two strongly conserved Tyr and His resi- in bacterial genomes. We confirm that it functions as a TA sys- dues. An alignment of the Sphingobium sp. YBL2 toxin with tem in E. coli, an organism with no native COG5654–COG5642 homologs from various pathogens illustrates that outside of these pairs, suggesting a target well-conserved in bacteria. We then report a and neighboring residues, there is little shared sequence similarity

high-resolution crystal structure of the protein complex, which in- (Fig. 1B). The function of the RES domain is unknown, and a BIOCHEMISTRY

Fig. 1. Bioinformatic analysis of the COG5654–COG5642 TA family. (A) Lasso peptide gene cluster containing the TA pair studied here (yblIJ). (B) Sequence similarity of the COG5654 toxin from Sphingobium sp. YBL2 and several bacterial pathogens. Residues with >70% similarity are shaded. Similarity is clustered around the strictly conserved Arg-31, Tyr-41, and Glu-52 residues. (C) Distribution of this TA family among bacterial phyla is shown at Left; Proteobacteria have by far the largest number of examples of this TA system. A further breakdown within Proteobacteria is shown at Right.

Piscotta et al. PNAS | January 15, 2019 | vol. 116 | no. 3 | 827 Downloaded by guest on October 2, 2021 BLAST of these toxins offers no homologous proteins with found in SI Appendix, Table S2 and Dataset S2, respectively. The known activities. COG5642 antitoxins meanwhile have a roughly TA family was not observed in any archaeal genomes, in agree- 60-aa C-terminal domain of unknown function, DUF2384, which ment with Makarova’s findings in 2009. The results of our search follows an N-terminal DNA binding domain. Antitoxins from confirm that the COG5654–COG5642 system is widespread in type II TA modules generally have C-terminal toxin binding bacteria, even more so than originally thought. domains, making this a likely function of DUF2384 (25). As Fig. 1B shows, COG5654 toxins appear in the genomes of The COG5654–COG5642 Pair Functions as a TA System in E. coli. To many pathogenic bacteria. To better understand the prevalence of more easily study the COG5654–COG5642 TA pair, we moved the COG5654–COG5642 system, we devised code (Dataset S1) the component genes from Sphingobium sp. YBL2 into E. coli. that searched all Refseq genomes for genes encoding an RES Although laboratory strains of E. coli harbor dozens of TA loci, domain protein downstream of a DUF2384 protein. The results of the COG5654–COG5642 pair is not one of them, and thus, our this search are summarized in Fig. 1C. Of the 108,787 bacterial first aim was to confirm that this system can still function in this Refseq genomes searched, there are 15,354 organisms that con- nonnative organism (10, 25). The toxin was expressed from a tain at least one copy of this TA family (14.1%) and 16,964 total low-copy pBAD33 plasmid (26), and upon induction, exerted a instances. Some organisms contained many copies, with Spirosoma bacteriostatic effect as measured by OD600 (Fig. 2A). We also spitsbergense and Spirosoma luteum having 12 and 10, re- examined cell viability by colony forming unit (cfu) count. One spectively. Sphingobium sp. YBL2 itself has five loci, three on hour after toxin induction, the cfu count had decreased dra- its chromosome and two on plasmids. An alignment of the five matically, although it began to increase at 3 h (SI Appendix, Fig. toxins reveals high identity (45%) and similarity (53%) between S1). This is similar to what was observed with RelE, a well-known two of the chromosomal toxins, but little between the remaining bacteriostatic toxin (27). Coexpression of the COG5642 antitoxin three, including the one we originally identified (SI Appendix, restored the normal growth phenotype, confirming that this Table S1). nonnative TA system functions normally in E. coli (Fig. 2A). To avoid overrepresentation of bacteria with many strains We then performed an alanine scan on the highly conserved characterized (e.g., M. tuberculosis or Bordetella pertussis), we con- residues of the RES domain described in the previous section. solidated each into a single species entry. After doing so, the TA Substitution of alanine for Arg-31, Glu-52, and His-56 resulted in family was found in 2,942 unique bacterial species of 16,826 the elimination of the toxic phenotype (Fig. 2B). Conversely, (17.5%). Within these, we found the COG5654–COG5642 pair in substitution of alanine for Tyr-41 and Ser-122 had no effect on B 15 bacterial phyla, although it was by far the most abundant and toxicity (Fig. 2 ). While this is surprising in both cases, it is less overrepresented in Proteobacteria, occurring in 2,527 of 7,483 spe- so for Ser-122, where the change is more conservative. Indeed, the alignment from Fig. 1B reveals that the corresponding amino cies (33.8%) (Table 1). The phyla also included Verrucomicrobia, acid in M. tuberculosis is itself alanine. We subsequently exam- Deinnococcus-Thermus, Spirochaetes, Planctomycetes, and ined the mildly conserved Ser-21, 44, and 45 to exhaust candi- Firmicutes, all of which were not observed earlier in Makarova dates for the namesake serine in the “RES” domain, but these et al. Within Bacteroidetes, we identified 254 species and noticed substitutions also had no effect on toxicity (SI Appendix, Fig. S2). a remarkably high overrepresentation in the Chitinophagia, To confirm that the loss of toxicity of the R31A, E52A, and Cytophagia, and Sphingobacteriia classes, occurring in 27/33 H56A toxins was not driven by low expression, we cloned these (81.8%), 90/115 (78.3%), and 45/65 (69.2%) organisms, respec- variants into high-copy pQE80 vectors. The R31A toxin expressed tively. There were 86 Actinobacteria identified, including 55 at a high level as judged by SDS/PAGE and no growth defect was Mycobacterial species. Curiously, of the 3,406 Firmicutes ge- observed, while the E52A toxin recovered the toxic phenotype, nomes, only a single instance (0.03%) of this TA family was likely due to the increased copy number of pQE80 relative to found in Exiguobacterium sp. S17, which may warrant further pBAD33 (SI Appendix, Fig. S3) (28). Neither the wild-type nor the investigation. Most Proteobacterial species were Alpha- (1,007/ H56A toxin could be cloned into pQE80. This suggests that there 2,244, 44.9%), Beta- (542/1,167, 46.4%), and Gammaproteo- is still residual toxicity of the H56A variant due to leaky expres- bacteria (949/3,617, 26.2%), with many from orders of known sion. Taken together, these results indicate that while Glu-52 and pathogens, such as Burkholderiales, Legionellales, and Pseudo- His-56 play a role in toxicity, Arg-31 is of paramount importance. monadales. A selected and full breakdown of the results can be Crystallography Reveals Structural Similarity to mARTs. With no reasonable starting point for elucidating the function of the Table 1. Breakdown of bacterial Phyla containing the – COG5654 toxin, we instead set our sights on determining its COG5654 COG5642 TA family structure, hoping to later infer its activity from this result. TA-bearing Unique Because the wild-type toxin can only be made at a high level species species searched Percentage, % Phylum when expressed alongside the cognate antitoxin, we set out to crystallize the protein complex first. For easily controlled 14 27 51.85 Acidobacteria coexpression, we cloned toxin and antitoxin into a pRSFduet 86 3,469 2.48 Actinobacteria vector. The complex was expressed for 3 h at 37 °C before native 254 1,334 19.04 Bacteroidetes purification, with typical yields of >5 mg/L. In our experimental 1 8 12.50 Balneolaeota setup, although only the toxin bears an affinity tag, the untagged 6 17 35.29 Chlorobi antitoxin copurifies due to strong protein–protein interactions 25 302 8.28 Cyanobacteria characteristic of TA systems (Fig. 2C) (29, 30). Furthermore, the 11 62 17.74 Deinococcus-Thermus proteins elute as a single peak during size exclusion chromatog- 1 3,406 0.03 Firmicutes raphy, with a retention time indicating a 2:2 stoichiometry (a TA 2 3 66.67 Gemmatimonadetes dimer) (SI Appendix,Fig.S4). 1 1 100 Tectomicrobia This purified TA complex had a solubility limit of 1 mg/mL 3 41 7.32 Planctomycetes and was unsuitable for crystallization. The general understanding 2,527 7,483 33.77 Proteobacteria of TA systems is that the antitoxin is inherently less stable than 1 5 20 Rhodothermaeota the toxin, demonstrated previously in vitro by the selective total 2 128 1.56 Spirochaetes degradation by trypsin of the VapB antitoxin from a folded 6 40 15 Verrucomicrobia VapBC complex (10, 31). We adapted this procedure for our

828 | www.pnas.org/cgi/doi/10.1073/pnas.1814633116 Piscotta et al. Downloaded by guest on October 2, 2021 Fig. 2. Heterologous expression in E. coli of the Sphingobium sp. YBL2 COG5654–COG5642 TA system. (A) Growth curves showing the effect of toxin expression with and without antitoxin induction 30 min prior. The toxin exerts a bacteriostatic effect that is prevented by antitoxin coexpression. (B) Growth curves of toxin variants with alanine substitutions for five highly conserved amino acids. R31A, E52A, and H56A variants are all nontoxic. Asterisks in A and B

indicate the timepoint of induction of toxin. (C) SDS/PAGE gel from a native purification of N-terminally His-tagged toxin coexpressed with untagged an- BIOCHEMISTRY titoxin. Its interaction with the toxin results in copurification despite the lack of a His tag.

system and observed by LC/MS that even after overnight trypsin places Arg-31 in a conformer directed away from the active site, treatment, a 72-aa C-terminal antitoxin fragment was intact de- also engaging it in hydrogen bonding with the toxin Ala- spite the existence of multiple possible cut sites (SI Appendix, 94 backbone oxygen. The strength of the TA interface is illus- Fig. S5). This, we hypothesized, was due to extensive TA contacts trated experimentally by urea-induced dissociation, where puri- making the remaining sites inaccessible. We constructed a series fied TA complex required incubation in 6 M urea to separate the of antitoxin truncations that left the C-terminal portion intact component proteins (SI Appendix, Fig. S8). and found that even the shortest antitoxin fragment we tested A significant interface also exists between the two toxin sub- (amino acids 99–159) could bind and neutralize toxin when units, with an interfacial surface area of 762.2 Å2 and ΔiG of coexpressed (SI Appendix, Fig. S6). Upon trypsin digestion, the −7.1 kcal/mol. The network of hydrogen bonding is less extensive, toxin lost only its two most C-terminal amino acids. This trypsin- but notably there is an interaction between the highly conserved treated complex exhibited solubility greater than 10 mg/mL and His-56 of one toxin subunit and Tyr-153 of the other (Fig. 3D). showed promise in crystal screening. Salt bridging between the toxin subunits occurs at Glu-127 and The trypsin-digested toxin contains only a single methionine Glu-128 of one subunit and Arg-46 and Arg-149 of the other (Fig. (Met) residue. To increase Met content for selenomethionine 3D). To determine if these salt bridges are necessary for toxicity, (SeMet) substitution and single-wavelength anomalous disper- we constructed a series of mutations at the two glutamate residues. sion (SAD), we used a L48M toxin variant, which we confirmed Single mutations to alanine had little effect on toxicity, but a retained toxicity (SI Appendix, Fig. S7). We successfully crystal- double mutation to even sterically similar glutamines rendered the lized the trypsin-treated SeMet-substituted L48M TA complex protein nontoxic (SI Appendix, Fig. S9). Thus, although a single and determined its structure to 1.55 Å using SAD (PDB ID code: pair of salt bridges can be disrupted, the presence of at least one is 6D0I). The complex crystallized in the P1 space group with unit critical for interfacial integrity and proper function. This strongly cell dimensions of 41.98 Å, 51.32 Å, and 57.94 Å. From this, we supports the idea of the toxin working as a dimer in vivo. Lastly, subsequently determined the structure of the trypsin-treated there is no discernable interface between the two antitoxin frag- wild-type complex to 1.50 Å (PDB ID code: 6D0H; Fig. 3A). ment subunits, suggesting these two exclusively interact with the A full description of data collection and refinement parameters toxins they bind. can be found in SI Appendix, Table S3. The complex crystallizes Following the elucidation of the complex structure, we employed as a dimer, where the C terminus of each antitoxin is buried the DALI structural similarity server to determine if any simi- in a putative toxin active site cleft housing all five highly con- larity to known proteins existed (33). The top result from this served residues discussed above (Fig. 3B). search was the catalytic domain of diphtheria toxin from We used PDBePISA to analyze the interactions between TA Corynebacterium diphtheriae, which had a Z-score of 7.0 and complex subunits (32). The surface area of the TA interface is rmsd of 3.0 Å, indicating some structural similarity despite only 1,369.6 Å2 with an average ΔiG (free energy gain on interface 11% sequence identity (SI Appendix, Fig. S10). Diphtheria toxin + formation, excluding hydrogen bonds and salt bridges) of is a secreted mART capable of cleaving NAD into nicotinamide −13.7 kcal/mol, indicating a strong affinity between the two and ADP-ribose. It transfers the latter to a posttranslationally proteins. Extensive hydrogen bonding occurs between 13 anti- modified histidine, diphthamide, in eukaryotic elongation factor- toxin and 14 toxin residues. Interestingly, salt bridging occurs 2 (EF-2), which inhibits protein synthesis (34). Other mARTs between the C-terminal carboxyl of the antitoxin and both the such as cholix toxin from Vibrio cholerae and exotoxin A from His-56 and critical Arg-31 of the toxin (Fig. 3C). This interaction Pseudomonas aeruginosa also exhibited strong Z-scores (SI Appendix,

Piscotta et al. PNAS | January 15, 2019 | vol. 116 | no. 3 | 829 Downloaded by guest on October 2, 2021 Fig. 3. Crystal structure of the trypsin-treated TA complex. (A) Overall structure of the TA dimer (toxin, orange; antitoxin, blue) determined to 1.50-Å resolution. (B) Close-up of the putative active site. The five highly conserved residues examined in the alanine scan are highlighted. (C) Bonding network of the critical toxin His-56 and Arg-31. The arginine sidechain forms salt bridges with the C-terminal carboxyl of the antitoxin, which establishes a rotamer that also results in hydrogen bonding with the toxin’s own Ala-94. (D) Structure of the toxin-toxin interface. Shown are the intersubunit salt bridges between Arg- 46 and Glu-128′ and Arg-149 and Glu-127′, as well as hydrogen bonding between Tyr-153 and the functionally important His-56′.

Table S4) (35, 36), and we hypothesized based on these results that observed that after 3 h, toxin ADP-ribosylation had greatly the COG5654 toxin is also a mART. Of note, the results also decreased relative to the wild type (SI Appendix,Fig.S12), contain several poly-ADP-ribosyltransferases (pARTs) including supporting the idea that the loss of toxicity of the R31A toxin is tankyrase-1 and -2, although these generally have lower Z-scores a result of a loss of transferase activity. The reactions were also + than the mARTs (Dataset S3). carried out with wild-type toxin using an NADP substrate, and we observed similar levels of glycohydrolase activity and automodifi- + Phosphoribosyl Pyrophosphate Synthetase Is a Putative Toxin Target. cation as for NAD (SI Appendix,Fig.S13A). This result was sur- + To test for mART activity, we required an isolated toxin sample. prising as mARTs generally show high specificity toward NAD . As described in the previous section, this could not be achieved Encouraged by these results, we sought to identify a cellular by a trypsin digestion due to the strong interactions between substrate for the toxin. We hypothesized based on toxin self– toxin and antitoxin. A typical procedure for toxin isolation for ADP-ribosylation that the target was a protein and not a many TA families involves complex denaturation, toxin repur- nucleic acid as was recently observed with DarT (19). Here, we ification, and refolding by dialysis (16, 29, 30). We adapted this employed a pulldown where cell lysates from toxin-expressing method for our system, noting that the toxin would only suc- cultures were incubated with macro domain protein AF1521 cessfully refold when arginine was present in the dialysis buffer from Archaeoglobus fulgidus and subject to His-tag affinity pu- (37). When dialysis was complete, arginine could then be removed rification. Macro domain proteins such as AF1521 have been without causing toxin aggregation. used previously to isolate ADP-ribosylated proteins from com- As a first test of mART activity, we incubated 1 μM toxin with plex mixtures such as cell lysates (43, 44). Several proteins were + 1 mM NAD . mARTs such as diphtheria toxin and iota toxin pulled down via this technique, and all were identified after an from Clostridium perfringens have been shown to exhibit weak in-gel tryptic digest, followed by LC/MS and Mascot peptide NADase activity in the absence of a protein target (38–40). We mass fingerprinting (SI Appendix, Fig. S14). These proteins, + observed not only a decrease in NAD and increase in free YfbG, GlmS, and EF-Tu, are all common contaminants associ- ADP-ribose, but also the appearance of an ADP-ribosylated ated with His-tag affinity purifications (45, 46). We were in- toxin (SI Appendix, Fig. S11). A tryptic digest revealed that this trigued, however, by the last protein we identified, phosphoribose modification occurred on a single peptide that contains the im- pyrophosphate synthetase (Prs), which is not a known affinity portant Glu-52 and His-56 residues (SI Appendix, Fig. S11C). purification contaminant. Prs is a homohexameric protein enco- Although we are unsure of its significance for this system, auto– ded by the essential prs gene, which is found in both bacteria and ADP-ribosylation is a known regulatory mechanism for several eukaryotes and strictly conserved in all nonparasitic organisms mARTs and may serve a similar function here, regulating toxin (47). Prs catalyzes the conversion of ribose 5-phosphate to phos- activity once disassociated from antitoxin in vivo (41, 42). We phoribose pyrophosphate, a precursor in the synthesis of nucleotides, + + repeated this reaction using the inactive R31A toxin variant and histidine and tryptophan, NAD ,andNADP (48). Its critical position

830 | www.pnas.org/cgi/doi/10.1073/pnas.1814633116 Piscotta et al. Downloaded by guest on October 2, 2021 in metabolism and strong conservation across a range of or- To confirm that these Prs modifications are not occurring ganisms make it a logical target for cellular toxins. nonspecifically, we used higher energy collision dissociation (HCD) LC/MS2 to analyze tryptic digests of the in vitro reactions The COG5654 Toxin ADP-ribosylates Prs in Vitro. We next sought to (50). Analysis with Scaffold proteomics software revealed that μ reconstitute Prs ADP-ribosylation in vitro. Reactions of 10 M the majority of ADP-ribosylation occurred at either Lys-182 or μ + Prs, 5 M toxin, and 10 mM NAD were set up at 37 °C, and the Ser-202 of the E. coli Prs, showing that these modifications are products were analyzed by LC/MS at 3 h and 20 h. After 3 h, specific (Fig. 4B and SI Appendix, Fig. S17). Lys-182 is conserved about one-third of the Prs was ADP-ribosylated (Fig. 4A). Al- among many bacterial Prs and has been shown to participate in though most of the modified Prs was singly ADP-ribosylated (∼25% of the total), a small amount of Prs had acquired a sec- ATP binding, placing it at the Prs active site (51). The Prs from ond ADP-ribose. When the reaction was run for 20 h, the amount Sphingobium sp. YBL2 has an aspartic acid (also a mART target) of ADP-ribosylated Prs had increased, with roughly equal at this position, which is also present in M. tuberculosis and other amounts of unmodified, singly modified, and doubly modified Prs bacteria, as well as in all three human Prs isozymes (51). A present (Fig. 4A). mutation of this residue to histidine in human isozyme one has Holding the concentration of Prs constant, we also performed been implicated in dysregulation in patients with Prs superac- + in vitro reactions at lower toxin (1 μM) and/or NAD (1 mM) tivity, suggesting that an ADP-ribose modification could also + concentrations. This concentration of NAD is below the phys- critically alter Prs in bacteria (52). Ser-202 shows some conser- iological level in E. coli (49). We were interested in both how this vation but, to our knowledge, is not critical for catalytic activity would affect the rate of reaction, as well as if these conditions or regulation. It is, however, part of a flexible loop involved in would still drive the formation of doubly ADP-ribosylated Prs. catalysis (53), and its modification could prevent this region from After 3 h, about 10–15% of Prs had been ADP-ribosylated (down entering a productive conformation. Although the corresponding from ∼33%) and little to no doubly modified Prs was present. valine in Sphingobium sp. YBL2 Prs cannot be ADP-ribosylated, Additionally, the effect was independent of whether toxin or + a targetable threonine is adjacent. Further investigation into NAD (or both) concentration had been lowered (SI Appendix, these modifications is necessary, both to determine if one is a Fig. S15). By increasing the reaction time to 20 h, singly modified preferred substrate and what effect the modification has on Prs peaks doubled in size, now accounting for ∼25% of total Prs. As + activity. Although we wished to repeat these reactions with the with high toxin and NAD concentration, doubly modified spe- Sphingobium sp. YBL2 Prs, the protein expressed poorly in E. cies showed the most increase from 3 h to 20 h, now present at BIOCHEMISTRY coli and coeluted with large quantities of GroEL (SI Appendix, roughly the same amount as singly modified Prs. This longer time ’ course suggests that the additional modification is not merely a Fig. S18). Based on the toxin s activity, we propose to rename the function of high reactant concentration. Also of note, we ob- RES domain family of toxins to ParT (Prs ADP-ribosylating served that the extent of toxin auto–ADP-ribosylation decreased toxin) and the corresponding antitoxin family to ParS. ’ + in the presence of Prs, supporting the idea that self–ADP- Noting ParT s ability to cleave NADP , we also attempted to ribosylation may be a form of regulation in the absence of target modify Prs with this substrate, but no pADP-ribosylation could (SI Appendix, Fig. S16). Collectively, this data shows that the be detected (SI Appendix, Fig. S13B). This agrees with what is toxin can ADP-ribosylate Prs twice, a surprising result given typically observed for mARTs and demonstrates that here, al- that mARTs generally ADP-ribosylate only a single amino acid though there may be some relaxed specificity for glycohydrolysis, + on their target. transferase activity still requires the traditional NAD substrate.

Fig. 4. In vitro ADP-ribosylation of E. coli Prs. (A) ESI mass spectrometry of the reaction of Prs (10 μM) with toxin (5 μM) and NAD+ (10 mM). After 3 h, conversion to a singly ADP-ribosylated Prs is ∼25%, with a doubly ADP-ribosylated peak beginning to appear. At 20 h, ∼65% of the Prs is ADP-ribosylated, with ∼35% singly modified and ∼30% doubly modified. (B) Crystal structure of the E. coli Prs hexamer (PDB ID code 4S2U). The ADP-ribosylated residues identified by LC/MS2, Lys-182 and Ser-202, are shown in gray. The Lys-182 sidechain is directed toward the Prs active site, while Ser-202 is located on a catalytic flexible loop.

Piscotta et al. PNAS | January 15, 2019 | vol. 116 | no. 3 | 831 Downloaded by guest on October 2, 2021 An Inactive Prs Variant Attenuates the ParT-Induced Growth Defect. To confirm that the change in OD600 observed in cultures To confirm that ParT targets Prs in vivo, we planned to sup- coexpressing toxin and H131A Prs was not due to cell morphology plement Prs on a plasmid. An analogous experiment with HipA changes or effects unrelated to ParT, we also examined cell via- demonstrated that overexpression of its target, GltX, allowed for bility. Cfu counts were taken immediately before ParT induction, recovery of normal cell growth (16). It is known, however, that as well as 15 min and 1 h after induction. Within 15 min, the vi- Prs hyperactivity is toxic to human cells (54), and indeed, we ability of E. coli expressing only ParT had fallen drastically by four observed a growth defect in E. coli when overexpressing the wild- orders of magnitude (Fig. 5 B and C). Cultures that had been type protein, making direct supplementation of Prs impossible expressing H131A Prs before ParT induction showed a single (SI Appendix, Fig. S19A). To overcome this issue, we instead order of magnitude decrease in cell viability, markedly better than supplied an inactive H131A variant of E. coli Prs, which alters a ParT-only cells. This result strongly agrees with the growth data histidine involved in binding the γ-phosphate of the ATP sub- and confirms that the increased cell density is due to improved strate (55, 56). As expected, overexpression of this H131A Prs viability caused by the presence of H131A Prs. caused no negative effects on cell growth (SI Appendix, Fig. S19B). Supplying an inactive Prs is not ideal, but we reasoned Discussion that the overexpressed protein could at act as a decoy, with ParT Here, we provide biochemical and structural insights into the na- targeting the abundant inactive Prs rather than the productive ture of the COG5654–COG5642 TA system, identifying the toxin native enzyme. Although eventually the native Prs would also get as a mART that targets the essential metabolic enzyme Prs. We modified, we believed this should at least attenuate ParT toxicity. have thus renamed the toxin parT and the cognate antitoxin parS. We expressed H131A Prs for 1 h before ParT induction. Rather We determined a high-resolution crystal structure of the ParST than the immediate growth arrest that is seen in cultures expressing complex, which revealed that the complex crystalizes as a dimer only ParT, cultures also expressing H131A Prs continued to grow (Fig. 3A). Disruption of the dimeric interface between the toxins following toxin induction (Fig. 5A). Although growth is slower than leads to a loss of the toxic phenotype (SI Appendix,Fig.S9), illus- in ParT-uninduced cultures, final OD600 is considerably higher in trating that ParT is a mART that functions as an obligate dimer. H131A Prs coexpressing cultures than in those expressing the toxin The C terminus of each ParS antitoxinisburiedintheactivesite alone (Fig. 5A). This result agrees with our hypothesis that although cleft of the cognate toxin, forming a salt bridge with both His- H131A Prs can mitigate toxicity, it cannot completely overcome it. 56 and Arg-31 of ParT. Among mARTs, this is an example with A similar effect was observed in cultures expressing H131A Prs for a high-affinity inhibitor that directly binds the mART active site. only 30 min before ParT induction, but to a lesser degree. Although a BLAST of ParT reveals sequence similarity only to To rule out the other proteins identified in the macro domain other RES domain proteins, a DALI search revealed multiple pulldown as purely contaminants and not possible ParT targets, mARTs with similarities in secondary structure, including exo- we repeated this coexpression with EF-Tu in place of Prs. We toxins such as diphtheria, cholera, and pertussis toxins (Dataset chose EF-Tu because of its association with multiple other post- S3). However, key differences emerge between ParT and these translational modifications, including phosphorylation, methylation, known bacterial mARTs, making it a unique addition to this and acetylation (57–59). Unlike in the case of Prs, expression of EF- family of enzymes. First, the exotoxins are all secreted and have Tu for 1 h before ParT induction led to a phenotype that was in- eukaryote-specific targets. ParT, however, acts in the cytoplasm distinguishable from cultures expressing ParT only (SI Appendix,Fig. of its producing cells and its target, Prs, is conserved among S20). This result strongly suggests that EF-Tu is not a target of ParT. all free-living organisms. The secretion of mART exotoxins is

Fig. 5. Coexpression of ParT with an inactive Prs variant suppresses ParT toxicity. (A) Growth curves of cells coexpressing H131A Prs and ParT. H131A Prs was

induced at 4 h (**) and toxin induced 1 h later (*). Cultures that accumulated H131A Prs showed a marked increase in OD600 following toxin induction. (B) Cell viability as measured by cfu count. Within 15 min, cfu count drops dramatically (∼10,000-fold) when cultures express only ParT. In contrast, cultures allowed to accumulate H131A Prs show only mildly decreased viability (∼10-fold). (C) Cfu count 15 min after toxin induction. Cultures were diluted 10,000-fold before 100 μL was plated on LB supplemented with the appropriate antibiotics and grown overnight at 37 °C.

832 | www.pnas.org/cgi/doi/10.1073/pnas.1814633116 Piscotta et al. Downloaded by guest on October 2, 2021 accomplished with an N-terminal signaling sequence, while up- Protein Expression and Purification. All His-tagged protein expression was take into eukaryotic cells is mediated in most cases through a induced in midlog phase BL21(DE3) E. coli (ΔslyD) and carried out for 3 h at distinct recognition domain. This is not the case for ParT, 37 °C. Proteins were purified via Ni-NTA native affinity purification according to the manufacturer’s protocol (Qiagen). For toxin isolation, TA complex however, which is comprised solely of the catalytic RES domain. was first purified as described above. The complex was dissociated in 6 M In this sense, ParT is most like C3 bacterial mARTs, which are urea, and denatured toxin was repurified by Ni-NTA affinity purification. also single-domain proteins, although they retain an N-terminal Arginine was then added to a final concentration of 200 mM, and the signal sequence (60). Furthermore, despite the similar domain sample was dialyzed against TBS supplemented with 200 mM arginine using architecture, C3 toxins (∼25 kDa) are substantially larger than a Slide-alyzer cassette (GE) followed by buffer exchange into TBS. ParT (∼18 kDa) and a structural alignment reveals little similarity. To our knowledge, ParT is the smallest example of a mART and, Growth Curves. Growth curves were measured using a Synergy 4 plate reader thus, may represent a mART that appeared early in evolution. (Biotek). In isolated toxin expression, expression was induced at the begin- The catalytic motif of ParT bears a resemblance to both diph- ning of exponential phase by the addition of arabinose. In coexpressions with other proteins, the nontoxic protein was induced 1 h before the toxin. Ex- theria toxin-like (DT) and cholera toxin-like (CT) mART motifs pressions were carried out for at least 4 h. but is distinct from both. The defining DT HYE motif features two highly conserved tyrosines, a histidine, and glutamate, all Trypsin Digestion of Native TA Complex. Trypsin digestion was carried out with critical for toxicity (60). ParT and other RES domain proteins sequencing grade modified trypsin according to the manufacturer’s protocol include each of these residues in their active site, with the Glu and without a denaturation step (Promega). The digested complex was analyzed His residues important for toxin function (Fig. 2B). ParT also by SDS/PAGE and LC/MS to determine the extent of degradation (LC: Agilent contains one conserved Tyr residue, but it is not critical for toxicity 1260 Infinity with Zorbax 300SB-C18 column, MS: Agilent 3560 Accurate- Mass qTOF). (Fig. 2B). The HYE motif of DT lacks an arginine residue com- parable to the critical Arg-31 residue in ParT (Figs. 2B and 3B), SeMet Protein Expression and Purification. Overnight cultures were used to highlighting the differences between these two mART active sites. inoculate M9 minimal media supplemented with 50 mg/L selenomethionine The CT RSE motif is defined by a critical arginine, an STS se- and 40 mg/L of all amino acids except for methionine. Cultures were grown to quence, and an (E/N)XE sequence (60). Despite being almost early log phase, at which point a solution of Ile, Leu, Val, Lys, Phe, and Thr identical in name to the RES domain in ParT, there are minimal (10 mg/mL each) was added to suppress Met biosynthesis. After 20 min, similarities between these active sites besides the critical arginine protein expression was induced. Expression, purification, and trypsin digestion of the complex were all carried out as described above. residue. Current structural and biochemical data on the mecha- BIOCHEMISTRY nism of mART exotoxins suggests that mART active sites stabilize + Protein Crystallization. A 10 mg/mL solution of trypsin-digested TA complex the cation formed upon glycohydrolysis of NAD (36, 61, 62). was subject to screening using a Phoenix crystallization robot (Art Robbins). Given that the ParT active site differs from both of the canonical On scaleup, crystals were grown at 20 °C using the sitting-drop vapor dif- mART active sites, it is an attractive target for further mechanistic fusion method. Drops were 1.5 μL:1 μL protein solution:MPD precipitant analysis of the ADP-ribosyltransferase reaction. solution (0.1 M sodium acetate trihydrate, 10 vol% MPD, pH 5.0). Crystals Recently, the DarT toxin was shown to ADP-ribosylate thy- typically reached maximum size in 2 wk, were harvested, and dipped in midines on single-stranded DNA, establishing nucleotidyl ADP- cryoprotectant (MPD solution and 10% glycerol) before vitrification. SeMet- ribosylation as yet another bacteriostatic mechanism of the di- substituted L48M TA complex was crystallized using identical methods. Data verse type II toxins (19). In contrast, our work demonstrates that for SeMet L48M TA complex was collected at the Brookhaven National Laboratory National Synchrotron Light Source II, and phasing was de- type II toxins can also be protein mARTs. Intracellular protein termined using SAD. The SeMet L48M TA structure was used to solve the mARTs in bacteria are exceptionally rare, with the only prior wild-type TA complex, with data collected in the Princeton macromolecular example a nitrogenase mART from Rhodospirillum rubrum (63). crystallography core facility with a Rigaku Micromax-007HF X-ray generator ParT exerts its bacteriostatic effect via modification of a cellular and Dectris Pilatus 3R 300K area detector. target, Prs. This represents an essential cellular function, nucleotide biosynthesis, targeted by TA systems. Further distinguishing the Macro Domain Pulldown and Prs Identification. E. coli expressing toxin for 1 h ParST system from DarTG is that ParS exhibits toxin neutraliza- were harvested and lysed by sonication. Separately, His-tagged macro tionviaaprotein–protein interaction, rather than the modification- domain protein was incubated with Ni-NTA slurry to saturate the resin and reduce nonspecific binding. The macro domain-slurry mixture was then reversing activity of ADP-ribosyl glycohydrolase DarG. While the incubated with the toxin-induced lysate for 2 h at room temperature be- structure of DarG was determined, there is no structure of DarT to fore purification. The elutions were analyzed by SDS/PAGE and subject to compare with the ParT structure. Furthermore, DarT and ParT an in-gel trypsin digest according to the manufacturer’s protocol (Thermo share little sequence similarity and DarT is 230 aa compared with Fisher Scientific). Digests were analyzed by LC/MS against the MASCOT the much smaller ParT (161 aa). A recent review article postulated protein database. that additional TA systems could harbor mARTs that modify pro- – teins (64). Our work here bears out that prediction and shows that In Vitro ADP Ribosylation. Reactions were performed in TBS at 37 °C. Prs concentration was held constant at 10 μM, while NAD(P)+ concentration was this type of TA system is exceptionally widespread across bacteria. varied between 1 mM and 10 mM and toxin between 1 μMand5μM. Methods Samples were taken after 3 h, and the reactions continued overnight (20 h total). For intact protein analysis, samples were injected directly onto the Plasmid Construction. All cloning was performed in E. coli XL1 Blue cells. Agilent LC/MS system described above. Trypsin-digested samples were Genes encoding toxin and antitoxin were amplified from the genome of used to determine the modification sites using a coupled Easy 1200 UPLC Sphingobium sp. YBL2. The organism was a gift from Xin Yan’s laboratory at and Fusion LUMOS mass spectrometer at the Princeton University core Nanjing Agriculture University, Nanjing, China. Antitoxin was cloned into high facility. copy pQE80-L, and toxin was cloned into low copy pBAD33. For coexpression, the proteins were cloned into pRSF-duet. Macro domain Cell Viability (Cfu Count). Cells transformed with toxin, toxin and blank pQE80-L, AF1521 as well as E. coli and Sphingobium sp. YBL2 Prs were cloned into or toxin and H131A Prs were grown to the beginning of log phase, at which pQE80-L. Mutations were introduced using PCR site-directed mutagene- point nontoxic proteins were allowed to express for 1 h before toxin ex- sis. A full list of primers and plasmids used in this study can be found in SI pression was then induced. Samples of culture were taken immediately Appendix,TablesS5andS6. before toxin induction, as well as 15 min and 1 h after induction. These were serial diluted in LB and plated on LB with the appropriate antibiotics. Plates COG5654–COG5642 Genome Search. The code used to search for COG5654– were incubated overnight at 37 °C and colonies were counted manually the COG5642 TA pairs can be found in Dataset S1 in SI Appendix. next day.

Piscotta et al. PNAS | January 15, 2019 | vol. 116 | no. 3 | 833 Downloaded by guest on October 2, 2021 ACKNOWLEDGMENTS. We thank Dr. Tharan Srikumar of the Princeton Univer- with data collection, and Jordan Ash of Ryan Adams’ laboratory at Princeton sity Mass Spectrometry Facility for assistance with HCD LC/MS2 experiments, the University for assistance with writing and implementing code for the ParST ge- staff of beamline AMX at the National Synchrotron Light Source II for assistance nome search. Funding was provided in part by NIH Grant GM107036 (to A.J.L.).

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