Disorder- and Dynamics-Based Regulatory Mechanisms in Toxin

Review pubs.acs.org/CR

Disorder- and Dynamics-Based Regulatory Mechanisms in Toxin− Antitoxin Modules Remy Loris*,†,‡ and Abel Garcia-Pino†,‡ †

Molecular Recognition Unit, Structural Biology Research Center, VIB, Pleinlaan 2, B-1050 Brussel, Belgium Structural Biology Brussels, Department of Biotechnology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium

‡

Notes Biographies Acknowledgments References

1. INTRODUCTION Intrinsic disorder in proteins has been studied in a large number of contexts in eukaryotes but has received less attention in prokaryotes. In this review, we show by using bacterial toxin−antitoxin modules as a paradigm that, while less prevalent than in eukaryotes, intrinsic disorder in proteins from prokaryotes can be functionally important and that this functionality is not restricted to simple folding upon binding transitions. Originally identified as plasmid-stabilizing entities, toxin− antitoxin (TA) modules are ubiquitous in the genomes of prokaryotes and archeae.1,2 Most commonly they constitute small operons that encode two genes. The downstream gene encodes for a toxic protein, while the upstream “antitoxin” gene protects the cell against this toxin. The antitoxin can either be an antisense RNA preventing expression of the toxin gene (type I module),3,4 a protein that inhibits the toxin by forming a noncovalent complex (type II module), or,5,6 as discovered recently, a folded RNA species that binds to and inhibits the toxin (type III module).7 In addition, type IV TA modules have been defined on the basis of the example of the inhibition of polymerization of bacterial cytoskeletal proteins by the toxin YeeV that is counteracted by the antitoxin YeeU, but without the formation of a complex between YeeU and YeeV.8 Finally, the GhoS/GhoT pair has been defined as a type V TA module, where the antitoxin GhoS is an RNase that degrades the mRNA of the toxin GhoT, thus preventing GhoT synthesis in the presence of the antitoxin.9 Type II modules, the most extensively studied group and the focus of this review, consist of many different families with distinct distributions and biochemical activities and with complex evolutionary relationships between them.2 They nevertheless share a number of general characteristics: (i) Toxin and antitoxin are small cytoplasmic proteins, typically less than 150 amino acids in length. (ii) When grafted on plasmids, TA operons contribute to plasmid stability by a mechanism of postsegregational killing. (iii) The toxin interferes with a basic functionality of the cell such as

CONTENTS 1. Introduction 2. Modular Architecture and the Origin of TA Modules 2.1. Architecture of Canonical Type II TA Modules 2.2. Noncanonical TA Modules: Variations on a Theme 2.3. Nature of the Intrinsic Disorder in TA Antitoxins 3. Role of Intrinsic Disorder in Turnover of Antitoxins 4. Antitoxins as Molecular Recognition Elements 4.1. Folding upon Binding 4.2. HUB Properties 5. Parallel Rejuvenation Mechanisms for ToxinMediated Gyrase and Ribosome Poisoning 5.1. Rejuvenation of CcdB-Poisoned Gyrase 5.2. RelE-Poisoned Ribosomes 5.3. Regulation of MazF-Mediated mRNA Cleavage 5.4. Segmental Binding as a General Motif in Antitoxins 6. Transcription Regulation 6.1. Conditional Cooperativity 6.2. Low to High Affinity Switch 6.3. Allosteric Communication Involving an Intrinsically Disordered Domain 6.4. Steric Exclusion 6.5. A Simplified Version of Conditional Cooperativity in TA Modules with a Single Operator Site 7. Conclusions Author Information Corresponding Author © 2014 American Chemical Society

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Special Issue: 2014 Intrinsically Disordered Proteins (IDPs) Received: November 14, 2013 Published: May 7, 2014 6933

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Table 1. Overview of the TA Modules Discussed in This Review TA family

organism

ccdAB mazEF mazEF mazEF mazEF

E. coli plasmid F E. coli E. coli plasmid R1 B. subtilis S. aureus

relBE yoeB/yef M yoeB/yef M higBA mqsRA prlF/yhaV a-relBE a-relBE parDE parDE paaR2−paaA2−parE2

E. coli E. coli M. tuberculosis E. coli plasmid Rts1 E. coli E. coli P. horikoshi M. jannaschii C. crescentus E. coli plasmid RK2 E. coli O157

phd/doc tasAB vbhTA

bacteriophage P1 Bacillus thuringiensis B. schoenbuchensis

vapBC vapBC f itAB

R. felis S. flexneri N. gonorrheae

pezAT ω−ε−ζ

S. pneumoniae S. pyogenes plasmid pSM19 035

hipBA

E. coli

yp_293189

R. eutropha

toxin

antitoxin

CcdB/MazF Superfamily CcdB CcdA MazF MazE Kid Kis YdcE YdcD MazF MazE RelE/ParE Superfamily RelB RelE YoeB YefM YoeB YefM HigB HigA MqsR MqsA PrlF YhaV RelE RelB RelE RelB ParE ParD ParE ParD ParE2 PaaA2 FIC Superfamily Doc Phd TasB TasA VbhT VbhA VapC Superfamily VapC VapB VapC VapB FitB FitA PezT/ζ Family PezT PezA ζ ε HipBA Family HipA HipB Fused Toxin−Antitoxins − −

toxin function gyrase poison mRNA cleavage mRNA cleavage mRNA cleavage mRNA cleavage ribosome poison ribosome poison ribosome poison ribosome poison ribosome poison ribosome poison ribosome poison ribosome poison gyrase poison gyrase poison unknown phosphorylation of EF-Tu unknown adenylylation of 80 kDa endogenous protein cleavage of initiator tRNAa cleavage of initiator tRNAa unknown phosphorylation of uridine diphosphate-N-acetylglucosamineb phosphorylation of uridine diphosphate-N-acetylglucosamine phosphorylation of glutamyl tRNA synthase unknown

Assumed but only proven for VapC from Salmonella enterica serovar Typhimurium LT2. bAssumed but only proven for ζ (PezT homologue) from plasmid pSM19035 from S. pyogenes a

replication or translation.10−18 (iv) The antitoxin has a short in vivo lifetime compared to the toxin and is specifically targeted by intracellular proteases, requiring constant replenishment.19−24 (v) The antitoxin neutralizes the toxin by forming a tight noncovalent complex with it. This usually involves an intrinsically disordered segment in the C-terminal half of the antitoxin. In many cases, toxin−antitoxin complexes have the potential to form complex oligomers.25−27 Their function often transcends from being mere reservoirs of neutralized toxin to become an important piece in the regulation of the TA operon (depending on the stoichiometry, the complex may or may not act as a repressor or derepressor). In this review, we focus on the role of intrinsic disorder in the functioning and regulation of type II toxin−antitoxin modules. An overview of the different TA modules that are discussed in this paper is given in Table 1. Although less prevalent than in eukaryotes, intrinsically disordered proteins (IDPs) and protein segments form an important part of the proteome of bacteria and archaea.28−32 Studies on the functionalities of IDPs in prokaryotes are largely lacking. The relevance of intrinsic disorder in TA antitoxins became apparent about a decade ago when nanobody-aided crystallization of Escherichia coli MazE showed that the C-terminal half of its polypeptide chain lacked structure.33 This property immediately provided a potential

explanation for its susceptibility to proteolytic degradation, short in vivo lifetime, and reluctance to crystallize in the absence of a partner (at that time, only the crystal structures of two TA toxins were known, F-plasmid CcdB and Kid from plasmid R1;34,35 there were no structures of antitoxins or toxin−antitoxin complexes). These three properties are shared among antitoxins in general, independent of the protein family to which they belong. Since then, a body of evidence has accumulated that links intrinsic disorder of TA antitoxins to their different in vivo functions and physicochemical properties. As such, they have become a paradigm for the unprecedented variety of roles of intrinsic disorder in the Pprokaryotic proteome.

2. MODULAR ARCHITECTURE AND THE ORIGIN OF TA MODULES TA antitoxins typically contain a significant amount of intrinsic disorder, with variable degrees of prestructuring. The main function of the IDP segments is folding-upon-binding, and this functionality is directly linked to the evolutionary origin of TA modules. Therefore, in order to understand the nature and functionality of intrinsic disorder in antitoxins, we first have to look at the architectures of type II TA modules and how they likely evolved. 6934

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2.1. Architecture of Canonical Type II TA Modules

the interaction of the toxin with the antitoxin regulatory element modulates the affinity between antitoxin and operator DNA. Thus, the architecture of the antitoxins is modular, and the corresponding modules are exchangeable. The latter was proven experimentally for different members of the Phd family of antitoxins and can be deduced from the existence of many families of TA modules that share a common toxin and regulatory element but differ in their DNA-binding antitoxin domain, e.g., phd/doc and tasAB, or vapBC and fitAB.2,37 The well-studied RelE superfamily of toxins is associated with a variety of antitoxins carrying different DNA-binding domains (Figure 3). Depending on the nature of the antitoxin, the relBE family (RHH DNA-binding motif) is extended to prlF/yhaV (AbrB-like DNA-binding domain), yoeB/yef M (Phd-like DNAbinding domain), higBA, and mqsRA (HTH motifs). Similar varieties in domain association are found for the other TA toxins module.2 This modular architecture probably reflects a series of illegitimate recombination events that are at the origin of TA modules. One may consider a regulatory segment breaking off from a toxin precursor and fusing to a common transcription regulator domain, thus creating a module with novel regulatory properties. The strongest evidence for such a scenario comes from the phd/doc module.38 Here the toxin Doc is a truncated FIC domain. The C-terminal intrinsically disordered segment of the antitoxin Phd binds as a kinked α-helix to a site on Doc which in regular (outside at TA context) FIC domains is occupied by an α-helix located either at their N- or a Ctermini.39 Thus, the Doc-binding segment of Phd complements the fold of Doc (Figure 4), and in the absence of this binding partner it remains disordered. Furthermore, the exposed hydrophobic residues of Doc at its Phd binding interface could be an evolutionary relic from such a α-helix-transfer event. In agreement with our evolutionary model for phd/doc, there are several other TA complexes where the N-terminus of the toxin-neutralizing domain of the antitoxin is located close to the C-terminus of the toxin. This is also the case for E. coli ccdAB, yoeB/yef M and mazEF, C. crescentus parDE, and Neisseria gonorrheae f itAB).40−43 This architecture is in agreement with a generalization of the gene fission/fusion scenario of phd/doc. However, it should be noted that, with the exception of FIC domains, no other natural TA fusion of this type has been observed until now and that some TA families such as VapBC and higBA are exceptions to this rule. This structural property has nevertheless been exploited in E. coli mazEF to construct a fusion leading to a permanently inactivated MazF protein.44 The relBE family presents an interesting variation where the Cterminal helix of the RelE toxin is physically displaced by an αhelix from the antitoxin in the toxin−antitoxin complex.45

In a canonical type II TA module, toxin and antitoxin genes form an operon with the antitoxin gene located upstream of the toxin gene (Figure 1A) Antitoxins typically consist of a DNAbinding domain joint to a regulatory element (Table 2 and Figure 2). The latter is usually intrinsically disordered when not bound to the toxin partner,5 although there are exceptions such as in the mqsRA module.36 This regulatory element binds to the corresponding toxin and neutralizes its activity. Reciprocally,

Figure 1. Gene organization of type II toxin−antitoxin modules. Panels A−E depict the different variations in gene organization and corresponding proteins and protein complexes. Toxins are colored red, the antitoxin DNA-binding domains are shown in yellow, and the antitoxin-neutralizing domains are in orange. This color scheme is used consistently in all figures throughout this paper. (A) “Classic” arrangement with the toxin gene being upstream of the antitoxin gene and the DNA-binding domain of the antitoxin being placed N-terminal to the neutralization domain. This organization is found in most type II TA modules, including the archetypal relBE, mazEF, ccdAB, parDE, and phd/doc. (B) Inverted arrangement as observed for higBA and mqsRA modules. Not only is the toxin gene located upstream of the antitoxin gene but the neutralizing domain of the antitoxin is now at its N-terminus. (C) Three-component module where the DNA-binding domain (here called “regulator”) and the neutralizing domain (here called “antitoxin”) are encoded as distinct proteins. Except for the splitting of the antitoxin gene, the organization follows that of the classic modules. (D) “Incomplete” TA modules lacking an antitoxin DNA-binding domain or regulator. Such arrangements have until now been observed only in Gram-positive bacteria (e.g., S. aureus mazEF) and archaea (e.g., Methanococcus jannaschii relBE). The S. aureus mazEF module is regulated via SigB. (E) yp_293189 from Ralstonia eutropha encodes a single protein that is an apparent fusion between a Phd-like antitoxin and a RelE-like toxin The color gradation from yellow to red is used to indicate that the limits of the antitoxin and toxin fragments are not well-defined, with the central part of the gene product aligning equally well with the N-terminus of YoeB toxins as with the C-terminus of Phd antitoxins.

2.2. Noncanonical TA Modules: Variations on a Theme

In further agreement with gene recombination events being at the core of TA modules’ evolution is the existence of four other noncanonical type II TA modules. The first consists of TA operons where the antitoxin gene is located downstream of the toxin gene.1,2 Examples are the higBA and mqsRA modules. Consistent with the recombination/segment transfer hypothesis, in such modules the location of the two antitoxin domains is swapped as well, the neutralizing domain being located Nterminal to the DNA-binding domain (Figure 1B). A second variation consists of TA modules with the antitoxin lacking a DNA-binding domain (Figure 1C). An example here 6935

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a

6936

RelB RelB ParD ParD PaaA2

M. tuberculosis E. coli plasmid Rts1 E. coli

E. coli

P. horikoshi

M. jannaschii C. crescentus E. coli plasmid RK2 E. coli O157

bacteriophage P1

B. thuringiensis

B. schoenbuchensis

R. felis

S. flexneri

N. gonorrheae S. pneumoniae

S. pyogenes plasmid pSM19 035

E. coli

R. eutropha

yoeB/yef M higBA mqsRA

prlF/yhaV

a-relBE

a-relBE parDE parDE paaR2−paaA2−parE2

phd/doc

tasAB

vbhTA

vapBC

vapBC

f itAB pezAT

ω−ε−ζ

hipBA

yp_293189

Predicted from the amino acid sequence.

YhaV

B. subtilis S. aureus E. coli E. coli

mazEF mazEF relBE yoeB/yef M

Phd-like domain

HipB −

AbrB-like domaina AbrB-like domaina RHH motif no structure available

AbrB-like domaina absent

paired β-strand RHH motif RHH motif HTH motif in separate PaaR2 regulator Phd-like domain

AbrB-like domaina leucine zipper?

Phd-like domain HTH motifa HTH motif

AbrB-like domain AbrB-like domaina RHH motif absent RHH motif Phd-like domain

RHH motif

All-α domain from separate ω regulator helix−turn−helix

ε

FitA PezA

VapB

VapB

VbhA

TasA

Phd

YefM HigA MqsA

YdcD MazE RelE YefM

MazE Kis

E. coli E. coli plasmid R1

mazEF mazEF

CcdA

antitoxin

E. coli plasmid F

organism

ccdAB

TA family

dimerization/ DNA-binding domain toxin-neutralizing domain in free state

helix−loop−helix−loop− extended helix−loop−extended helix−loop−helix no structure available no structure available

two long helices no structure available helix−loop−strand helix−loop−helix−loop− extended no structure available no structure available folded Zn-containing α+β domain no structure available

kinked helix followed by short helix extended with short helix no structure available

toxin-neutralizing domain in bound state

not present

unknown

likely folded all-α domain

unknown likely folded three-helix bundle domain

unknown

unknown

unknown

helix−loop−extended as part of an all-α fold not present

helix−loop−extended helix−loop−helix as part of folded three-helix bundle domain long straight helix as part of folded all-α domain

helix−loop−extended

helix−loop−helix−loop− helix helix−loop−extended

disorder depends on ionic strength; at high ionic strength, the protein becomes unstructured from kinked helix Lys68 onward, and at low ionic strengths, the entire neutralization domain, as well as part of the DNA-binding domain, is disordered unknown no structure available

unknown unknown α-helix between Ala42 and Leu49; entirely disordered C-terminal from Leu49 two fluctuating stretches of α-helix embedded in otherwise unstructured polypeptide

unknown

unknown

ordered in α-helical segments in crystal structure unknown folded Zn-containing α+β domain

unknown unknown random coil NMR spectrum disordered after Leu64

no residual secondary structure single helical turn within otherwise unstructured domain

no residual secondary structure, but two ensembles differing in compactness

Table 2. Overview of the Antitoxins and Their Structural Properties Discussed in This Review

Chemical Reviews Review

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Figure 3. The RelE/ParE superfamily. All RelE/ParE superfamily members share the same microbial RNase fold first described for RNase T1 and barnase and, in most cases, also a conserved way of interacting with the antitoxin, but they display a variety of DNAbinding domains. (A) E. coli RelE interacts with RelB via its C-terminal domain that wraps around RelE via an α-helix and an extended segment. The N-terminal DNA-binding domain of RelB adopts a common RHH fold. (B) In the yoeB/yef m modules (here from E. coli), a similar interaction between toxin and antitoxin is observed as in relBE modules, but the DNA-binding domain of YefM adopts a Phd/ YefM fold. (C) A similar architecture is observed in the Caulobacter crescentus ParD−ParE complex, but with a helix−turn−helix motif in the DNA-binding domain of ParD. (D) The mqsRA modules (here again from E. coli) form an outlier within the RelE/ParE superfamily in the sense that the toxin-binding domain of MqsA is folded and its interaction with MqsR is unrelated to the other toxin−antitoxin interactions within the superfamily. The DNA-binding domain of MqsA forms a helix−turn−helix motif. (E) In the archeal Pyrococcus horikoshii OT3 RelB−RelE complex, again a “classic” RelE−RelB association is observed, but the neutralizing domain of RelB is preceded by a single α-helix. The latter in the crystal dimerizes to form a leucine zipper-like architecture, which was speculated to be a DNAbinding motif. (F) In the archeal M. jannaschii RelB−RelE complex, no DNA-binding domain is present in the RelB antitoxin, which otherwise interacts in a “classic” way with RelE. The color scheme is as in Figure 1. Each time a single toxin monomer is shown in red associated with an antitoxin neutralization domain in orange and its DNA-binding domain in yellow. DNA-binding domains, if present, are shown in their dimeric forms, but the neutralizing domain of the second monomer and also its associated toxin if present are omitted for clarity.

Figure 2. Folding-upon-binding and allosteric communication in antitoxins. (A) The antitoxin Phd of the P1 phd/doc module is largely disordered in its isolated state. The N-terminal domain shows partial structure at low salt concentrations, while its C-terminal toxinneutralizing domain remains fully unstructured. Binding of Doc to the neutralization domain of Phd not only leads to structuring of the latter domain but also of the DNA-binding domain of Phd. Mutational analysis of bacteriophage P1 Phd suggested that the α-helix induced by Doc in residues Ser40−Ala50 of Phd propagates toward the DNAbinding domain of Phd, shifting the equilibrium to its fully folded state and thus enhancing operator affinity. The resulting Doc−Phd2−Doc complex was shown by SAXS to be a very rigid entity. (B) The E. coli antitoxin MazE consists of an AbrB-type DNA-binding domain followed by an intrinsically disordered MazF-neutralizing domain. Upon binding MazF, the neutralization domain folds into a rather extended structure. Here the AbrB and neutralization domains of the antitoxin do not communicate directly. Rather, the folding and binding of the neutralization on MazF forces the latter to contact the AbrB domain as well. This direct stabilization of the AbrB domain through contacts with MazF is also expected to affect operator binding via allostery, although this has not been verified experimentally yet. (C) Although adopting different folds, the architecture of the RelB−RelE association is similar to that of MazE−MazF. The C-terminal domain of RelB is disordered in its isolated state, while its ribbon−helix−helix (RHH) DNA-binding domain is ordered in solution. The C-terminal IDP domain of RelB folds around RelE in a largely α-helical conformation, and again this leads to direct interaction between RelE and the RelB RHH domain and therefore also to further stabilization of the latter. Color scheme as in Figure 1

Three-component TA modules define the third noncanonical subgroup. They consist of a toxin gene preceded by an antitoxin gene that, as in the S. aureus MazE, has only toxinneutralizing activity (Figure 1D). Regulation of the operon involves a third regulator gene upstream of the antitoxin. The archetype of such a three-component TA module is the ω−ε−ζ module on plasmid pSM19035 from Streptococcus pyogenes,49 which is related to the classic two-component pezAT modules found in many Gram-positive genomes.50 Two different genes encode for the toxin-neutralizing activity (ε protein) and the DNA-binding activity (ω protein, RHH fold), in agreement with an independent origin of both domains. Also the parDE family has recently been extended with three-component versions on the E. coli O157 chromosome.51 Finally, yp_293189 from Ralstonia eutropha encodes a single protein that is an apparent fusion between a Phd-like antitoxin and a RelE-like toxin (Figure 1E). Both toxin and antitoxin

is the mazEF module from Staphylococcus aureus.46 The MazF toxin is a classic mRNA interferase with around 70% sequence identity to YdcE, a confirmed MazF protein from Bacillus subtilis.47 The corresponding antitoxin on the other hand is unexpectedly small and can be aligned only with the C-terminal half of the B. subtilis MazE-like antitoxin YdcD. Transcription regulation of the S. aureus mazEF module does not occur via a toxin−antitoxin complex. Rather, both genes are cotranscribed with the sigB operon.48 Another likely example of this type of module are the archeal relBE modules, where the antitoxin is expected to be entirely disordered in the absence of the toxin and where no obvious DNA-binding domain can be deduced from the crystal structure of the complex. 6937

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The degree of disorder observed in antitoxins varies and is listed systematically in Table 2 for the different antitoxins listed in this review. For F-plasmid CcdA, the C-terminal domain is essentially disordered with no residual secondary structure.52 Still, two conformational ensembles can be distinguished. Of these two ensembles, the minor one is more compact, shows decreased mobility for residues Val53−Glu68, and folds back onto the structured N-terminal domain. Crucial here are transient hydrophobic interactions within the segment Val53− Arg57 and between segments Ala66-Asp67 and Tyr20 and Leu39. Kis, a MazE homologue encoded on plasmid R1 shows a single α-helical turn for residues Leu56−Leu59, located in a region that in E. coli MazE adopts a two-turn α-helix when bound to MazF.26 As this result is based on secondary structure prediction by TALOS in the absence of a fully calculated structure, further information on the conformational ensemble of the C-terminal domain of Kis is still lacking. In E. coli RelB, the NMR spectrum of the isolated C-terminal domain (Lys47−Leu79) is characteristic of random coil.53 Analysis of its structural properties in the context of the full length protein, however, is difficult, as most resonances are broadened due to oligomerization. The C-terminal eight amino acids (Val72−Leu79) nevertheless show very sharp resonances, indicating a high degree of disorder. Residues Leu66−Lys70 were pinpointed as the culprit for aggregation, and a truncated version of RelE lacking residues Leu66−Leu79 showed the segment Arg43−Arg65 to be largely unstructured with low hNOE and random CSI values. In the Phd/YefM family, on the other hand, α-helix propensity in the C-terminal domain seems to be more pronounced. Both crystal structures of bacteriophage P1 Phd and Mycobacterium tuberculosis YefM show largely ordered structures for the C-termini of these two structurally related proteins.56,59 In the case of YefM, the C-terminal domain (Arg54−Glu91) consists of two α-helices connected via a short loop. This led the authors to conclude that M. tuberculosis YefM does not contain any natively unfolded region, in contrast to what was suggested for E. coli YefM based on the observations of the biophysical properties of the protein in solution.60 Given the low solvent contents of the crystals concerned and that the relative positions of the two C-terminal helices in the crystal structure of M. tuberculosis YefM differ significantly among the four copies in the asymmetric unit, it is likely that crystal packing contributes significantly to the observed conformations and that the protein may be less ordered in solution. Indeed, in the crystal structure of the E. coli YoeB−YefM complex, where one of the YefM monomers is not involved in interactions with YoeB, no electron density is observed beyond Leu64. Solution studies on M. tuberculosis YefM are unfortunately lacking, leaving the issue unresolved. An interesting twist is observed for Phd from bacteriophage P1, which belongs to the same superfamily of transcription factors as YefM. In the same crystal form of Phd, molecules with distinct conformations are observed.56 One Phd dimer is highly ordered with continuous electron density between residues Met1 and Asn67, leaving only Lys68−Arg73 unstructured. The second Phd dimer, however, is highly disordered. Not only is there no electron density observed for the Doc-neutralizing segment starting from residue Ala50, but the N-terminal DNA-binding domain is also much less ordered. The three N-terminal α-helices unwind to loop structures and become partially invisible in the electron density. The only

Figure 4. The FIC superfamily. The FIC superfamily consists of a domain consisting of six α-helices and a conserved active site loop. These domains often display an AMPylation activity, although many exceptions are known. FIC domains can exist as individual proteins or as domain of larger multidomain proteins. Three distinct FIC families can be discerned, depending on their mechanism of inhibition. (A) When part of a phd/doc TA module, the activity of the FIC domain is inhibited by an additional α-helix located at the C-terminus of the corresponding antitoxin. (B) In classic FIC domain, the regulatory helix is part of the FIC domain itself and is placed at its N-terminus, as in N. meningitidis FIC. (C) Alternatively, the regulatory helix is located at the C-terminus of the DIC domain, as in the VopS AMPylator from Vibrio parahemolyticus. The color scheme is as in Figure 1, except that the active site loops are depicted in light blue.

domains are truncated, with the Phd-like domain lacking most of the C-terminal intrinsically disordered segment. The RelElike domain lacks its N-terminal α-helix. Homology modeling suggests that the C-terminal α-helix of the Phd domain is likely to pack onto the β-sheet of the RelE domain, thus complementing its fold. The resulting putative protein does not seem to have retained its catalytic residues and would also not fit in the RelE binding site of the ribosome for steric reasons. Therefore, the functionality of this rare fused module, if any, remains to be identified and in all likelihood corresponds to an evolutionary dead end. 2.3. Nature of the Intrinsic Disorder in TA Antitoxins

Intrinsic disorder seems to be a recurring theme in TA antitoxins and particularly in their toxin-neutralizing domain. Experimental evidence for native state disorder and folding upon binding is available for the ccdAB, relBE, mazEF, parDE, yoeB/yef M, and phd/doc families,33,52−57 while for the archeal relBE family the structure of a toxin−antitoxin complex suggests that the antitoxin is unlikely to fold in the absence of the toxin partner.58 6938

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secondary structure element retained is the β-sheet, through which the Phd dimer is formed. The existence of the ensemble of ordered and disordered Phd dimers can be observed in solution by NMR spectroscopy. The equilibrium between both conformational states is strongly dependent on the external conditions, in particular ionic strength. These structural features are further reflected in the very low thermodynamic stability of Phd and its relatively weak cooperative unfolding, akin to a molten globule.56,61 For PaaA2, the structure of the ParE2-bound state of the antitoxin from the three-component paaR2−paaA2−parE2 module on the chromosome of E. coli O157 is not known.51 However, its structural ensemble in the unbound state has been characterized in detail by using a combination of SAXS and NMR spectroscopy.57 The protein, which lacks a DNA-binding domain, is highly disordered but contains two segments that adopt transient α-helical conformations. Although the two helices move relative to each other, the resulting conformational ensemble of the protein remains quite compact. Most likely, the observed prestructuring provides initiation points for the recognition of PaaA2 by ParE2 and will influence the affinity of PaaA2 for ParE2.

affinity between CcdA and CcdB is around 20 pM.40,69 Thus, when CcdB is activated through Lon-mediated degradation of CcdA, active ATP-driven unfolding of CcdA from CcdB is likely to be required.19,20 In the case of a pezAT module from Streptococcus pneumoniae, the PezT toxin remains inactive after inhibition of PezAT expression.70 PezA and PezT interact with femtomolar affinity, and as a consequence, complex formation is quasi-irreversible. This makes the PezA C-terminal domain resistant to proteolysis in its bound state, and the Lon machinery is not capable of removing PezA from PezT.

4. ANTITOXINS AS MOLECULAR RECOGNITION ELEMENTS The best known function of IDP segments is folding upon binding. In larger IDP segments found in eukaryoric proteins, this is often achieved via relatively short (10−70 amino acids) segments that often have high α-helical or β-strand propensities. Such segments have been termed molecular recognition elements (MoREs).71,72 The intrinsically disordered toxin-neutralizing regions of TA antitoxins contain properties of such MoREs, although they are not embedded in a larger “sea” of intrinsic disorder.

3. ROLE OF INTRINSIC DISORDER IN TURNOVER OF ANTITOXINS Antitoxins require a high turnover and thus rapid proteolitic degradation. Proteolysis of the neutralizing antitoxin is the key step in TA toxin activation, and thus intrinsic disorder may help reduce the in vivo lifetime of antitoxins relative to their cognate toxins. Prior to any structural knowledge on TA antitoxins, their rapid proteolytic turnover was originally linked to their low thermodynamic stabilities relative to their long-lived toxin partners.25,60,62,63 However, this link turned out to be poor, as several antitoxins turn out to have a quite significant thermodynamic stability, which we now know is due to their folded DNA-binding domain.53,64,65 The first structure of the antitoxin E. coli MazE showed the presence of a folded Nterminal domain and lack of electron density for the C-terminal half.33 This highly disordered protein was crystallized by means of a dromedary antibody-derived nanobody, and the corresponding structure immediately suggested that intrinsic disorder in antitoxins might be the key to their susceptibility to proteolytic degradation and the requirement for a rapid turnover compared to their cognate toxins.19,21,22,63 Direct evidence for the requirement of an intrinsically disordered segment to allow antitoxin degradation comes from HipB, where it was shown that a short 16 residue disordered Cterminal segment acts as a signal for Lon-mediated degradation.66 In contrast to other TA modules, this short IDP segment is not involved in toxin recognition or operator binding. Therefore, it is reminiscent of the intrinsically disordered initiator regions required for proteolytic degradation by the 20S and 26S proteasome.67 Recent evidence suggests that while this may be the case for some antitoxins, tuning of antitoxin lifetime may not be the key reason for the presence of intrinsic disorder. In the mqsRA module, the antitoxin MqsA interacts with the toxin MqsR in a well-folded domain36 yet is prone to proteolytic degradation by Lon.68 Furthermore, the very high affinities that are often observed between toxin and antitoxin are at odds with the antitoxin being presented in an unfolded state to the proteolytic machinery. For example, in the F-plasmid ccdAB module, the

4.1. Folding upon Binding

While not embedded in a larger IDP region, disordered toxinneutralizing regions carry the properties of typical MoREs, including their prediction as having a high α-helical content. An important functionality attributed to molecular recognition elements is that their folding-induced binding uncouples affinity and specificity.73 While the molecular mechanisms of coupled folding and binding have been the subject of intense research,74,75 the thermodynamic aspects have been mostly the subject of speculation. The thermodynamics of coupled folding and binding and its implications for uncoupling affinity and specificity were recently investigated for the C-terminal region of F-plasmid CcdA (CcdA37−72).69 The affinity of CcdA37−72 for CcdB stems from a combination of favorable intermolecular contacts and a highly unfavorable entropy of folding that is largely but not sufficiently compensated by a favorable enthalpy of α-helix formation. Mutations of both contacting and noncontacting residues modulate bindinginduced folding of CcdA37−72. The noncontacting mutants likely influence binding by altering the propensity of CcdA37−72 to adopt an α-helical structure in the unbound state. Early literature suggested that folding upon binding, in general, should lead to medium to low affinity interactions.73 This is definitely not the case for the CcdA−CcdB interaction with a dissociation constant in the picomolar range. Similar and even higher affinities are observed for other toxin−antitoxin pairs. The combination of a large and highly complementary contact interface, as well as favorable intramolecular interactions, is thus capable of offsetting the large entropic cost of coupled folding and binding. It is remarkable that during this process CcdA37−72 adopts a unique conformation that does not depend on the exact details of the surface with which it interacts. Indeed, F-plasmid CcdA37−72 adopts the same conformation when bound to its native F-plasmid CcdB partner or to the Vibrio fischeri CcdB homologue,69 where 9 out of the 21 residues on the CcdA-binding interface are substituted.76 Curiously, F-plasmid CcdB itself undergoes local conformational changes upon binding of CcdA.40 This suggests a narrow energy well for the folded conformation of 6939

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CcdA37−72, where the depth of the well is determined by the interactions between CcdA37−72 and CcdB, but its width is defined mainly by intramolecular interactions within CcdA37−72. 4.2. HUB Properties

A second property attributed to MoREs is their involvement as HUB regions capable of interacting with multiple partners, a feat achieved via their malleability in different conformations.77 This functionality at first glance contradicts achieving high specificity. The toxin-neutralizing domains of several antitoxins nevertheless do show some limited hublike properties: they are capable of recognizing two distinct binding sites on their toxin partner, and to achieve this they have to adopt two distinct conformations. This was first observed again for F-plasmid CcdA-CcdB, where the two binding sites partially overlap.40 The first high-affinity binding event leads to folding and binding of residues Ala41−Trp72 of CcdA. The second binding event still allows residues Ala41−Asn62 to fold and bind, but not the C-terminal segment Gly63−Trp72. The partial folding and binding leads to a highly reduced affinity for this second interaction. Although not experimentally verified, the crystal structure of the B. subtilis YdcD−YdcE complex78 suggests that a similar situation is likely encountered in the mazEF modules, where the fold of the MazF toxin strongly resembles that of CcdB.35,79,80 In phd/doc, the two binding sites are distinct but still differ in affinity by more than 2 orders of magnitude. In both sites, the C-terminal segment of Phd adopts an α-helical conformation, but with different length and mode of bending.56

5. PARALLEL REJUVENATION MECHANISMS FOR TOXIN-MEDIATED GYRASE AND RIBOSOME POISONING The roles of disorder that were discussed until now remained quite basic and do not tip on the more complex disorder-driven mechanisms associated with IDPs from eukaryotes. Recent data nevertheless indicates that intrinsic disorder in TA modules plays a more active role when it comes to silencing of previously activated toxins. This functionality of intrinsic disorder is best characterized in the ccdAB module40 and likely has parallels in other modules that target different aspects of cellular physiology. 5.1. Rejuvenation of CcdB-Poisoned Gyrase

In the ccdAB module, intrinsic disorder in the C-terminal antitoxin domain was shown to be crucial for its ability to rejuvenate CcdB-poisoned gyrase complexes.40 The binding site for CcdA is largely inaccessible in the CcdB−gyrase complex. The off-rate of the complex is very slow, making a simple competition strategy for CcdA-mediated rejuvenation of CcdBpoisoned gyrase complexes highly inefficient. Yet, this rejuvenation action occurs on a fast time scale, indicating a more complex mechanism. This mechanism was recently elucidated and involves the intrusion of the C-terminal eight amino acids of CcdA in the gyrase−CcdB complex (Figure 5A). Initial binding of this short segment induces a conformational change in CcdB, unlocking the CcdB−gyrase embrace. After dissociation of the CcdB−gyrase complex, the remaining part of the intrinsically disordered C-terminal domain of CcdA locks itself around CcdB, establishing a high-affinity complex. CcdB-mediated gyrase rejuvenation is an allosteric process. Initial binding of the C-terminal eight amino acids occurs at a site on the surface of CcdB distinct from the gyrase contact interface and leads to structural and dynamic changes in CcdB. As a consequence, CcdB can exist in two structural states: one

Figure 5. Parallels in intrinsic disorder-mediated rejuvenation mechanisms. (A) Reaction cycle of CcdB. CcdB binds to gyrase in its open conformation when it is ready for strand passage. This complex is very stable but can be resolved by CcdA. CcdA can reach its binding site on CcdB when the latter is bound on gyrase only via the C-terminal eight amino acids of CcdA, the remainder of the binding site being sterically inaccessible. Binding of this C-terminus of CcdA to CcdB causes a conformational change in CcdB that unlocks it from gyrase. The resulting ternary gyrase−CcdB−CcdA complex is only transient and has not been observed experimentally yet. During this release of CcdB, the C-terminal neutralization domain of CcdA further wraps around CcdB, resulting in tight (pM) binding, 6940

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ribosomes.81 Like F-plasmid CcdB, E. coli RelE is subject to a conformational change between an inactive RelB-bound state45 and an active ribosome-bound state81 (the latter essentially identical to its isolated state). Like CcdB, the RelB site is largely inaccessible in the RelE−ribosome complex, except to a short C-terminal segment of RelB. Therefore, it is possible that the binding of the C-terminus of RelB allosterically unlocks RelE from the ribosome (Figure 5B). This event would then be followed by the stabilization of the RelE−RelB complex, trapped in a high-affinity interaction state through the foldingupon-binding to RelE of an additional RelB α-helix. In the latter state, the C-terminal α-helix of RelE is displaced by an α-helix from the antitoxin, disabling its catalytic power toward RNA. It should be noted that this mechanism, though plausible, requires further experimental support. The off-rate of RelE for the ribosome needs to be determined, and the proposed rejuvenation mechanism would only make sense if this offrate is sufficiently slow.

Figure 5. continued preventing CcdB from attacking gyrase again. (B) Possible reaction cycle of RelE. RelE binds to the A site of the ribosome, where it can cleave mRNA that is being translated. In this ribosome−RelE complex, the binding site of RelE for RelB is again partially inaccessible, except for the very C-terminus of RelB. Thus, the C-terminus of RelB may intrude on the RelE−ribosome complex and trigger its release. During this release, RelB further wraps around RelE while inducing a conformational change. (C) Possible reaction cycle of MazF. MazF is structurally closely related to CcdB but binds and cleaves mRNA at a site distinct from the gyrase binding site of CcdB. Only a single mRNA is processed at once, although the mechanism that excludes binding of a second mRNA to the symmetric MazF dimer remains elusive. The C-terminal neutralization domain of MazE wraps around MazF in a way very similar to the CcdB−CcdA interaction, and like the ccdAB module, the MazF dimer can bind two MazE neutralization domains on partly overlapping binding sites with distinct high and low affinities. High-affinity binding of the first MazE neutralization domain is unlikely to inhibit mRNA binding and cleavage, at least not via simple competition for the same binding site. The latter is only accomplished via binding of the second MazE neutralization domain in what then becomes the low-affinity site. In this figure, toxins are drawn in red, antitoxins in yellow, the A subunit of gyrase and the 30S subunit of the ribosome in orange, and the B-subunit of gyrase and the 50S ribosomal subunit in green. Transient states are shown in lighter colors.

5.3. Regulation of MazF-Mediated mRNA Cleavage

MazF proteins are structurally related to CcdB proteins35 despite exhibiting a different biochemical activity: selected cleavage of mRNA11,12 and rRNA.15 The conservation not only of fold and topology but also of neutralization by the antitoxin suggests that both toxin families share a common ancestor, despite lack of clear sequence similarities (Figure 6A,B). The neutralization domain again consists of two distinct regions, one which sterically overlaps with the RNA target. MazF proteins seem to shuttle as well between two distinct structural states: catalytically active substrate-bound and catalytically inactive MazE-bound. The distinction between the two states resides mainly in the conformation of two loops

compatible with gyrase binding but incompatible with CcdA binding and one compatible with CcdA binding but incompatible with gyrase binding (Figure 5A).40 5.2. RelE-Poisoned Ribosomes

The disorder-dependent rejuvenation mechanism of CcdA may have a parallel in RelB-mediated rescue of RelE-poisoned

Figure 6. Segmental binding and inhibition of toxin activity. (A) B. subtilis MazEF (YdcD−YdcE) complex. The C-terminal neutralization domain of MazE (YdcD) wraps around the MazE (YdcE) dimer (shown in two shades of red) in an α-helical conformation and can be divided into an Nterminal segment (yellow) that sterically interferes with the RNA substrate (blue sticks) and a C-terminal segment (orange) that is located outside the substrate binding groove. (B) Equivalent view of the F-plasmid CcdAB complex. The CcdB dimer (red) adopts the same fold as MazF, as does the C-terminal domain of CcdA. The latter can again be divided in an N-terminal segment (yellow) that sterically hinders gyrase binding and a Cterminal segment (orange) that acts via allostery. In both MazF and CcdB, there are two binding sites for the N-terminal segments of the antitoxin neutralization domain. On the other hand, only one C-terminal segment can bind to either MazF or CcdB dimer due to steric overlap of their binding sites. (C) Segmental binding of Phd to Doc. Here the C-terminal segment of the neutralization domain of Phd (orange) sterically interferes with binding of the ATP substrate (blue) to Doc (red). The N-terminal segment of the neutralization domain of Phd (yellow) does not directly interfere with substrate binding (neither ATP nor EF-Tu). 6941

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Figure 7. Transcription regulation by conditional cooperativity. Transcription of most TA modules is regulated via a mechanism termed “conditional cooperativity”, where the toxin acts as a corepressor at low (typically 1:1) toxin:antitoxin ratios but as a derepressor when there is an excess of toxin. Different molecular mechanisms are used in different TA modules to achieve this goal, employing steric exclusion, a low to high affinity switch and allostery. (A) ccdAB. Affinity of F-plasmid CcdA for different operator sites (the F-plasmid ccdAB operator contains eight binding sites for CcdA dimers) is enhanced though bridging of CcdA dimers by CcdB dimers, thus creating an avidity effect. Direct contact between CcdB and the Nterminal domain of CcdA may further enhance affinity. In this alternating CcdA−CcdB array, both high- and low-affinity associations occur via the overlapping binding sites on the CcdB dimer for the intrinsically disordered C-terminal domain of CcdA. Excess of CcdB will exchange the lowaffinity contacts for high-affinity contacts, leading to soluble CcdB2−CcdA2−CcdB2 heterohexameric complexes. These complexes are rigid and cannot occupy adjacent sites on the ccdAB operator. (B) phd/doc. The mechanisms of conditional cooperativity in phd/doc largely parallels that within ccdAB, except that there are only two Phd binding sites on the phd/doc operator and that the high- and low-affinity sites for Phd are distinct sites on a Doc monomer. In addition, allosteric communication between the N- and C-terminal domains of Phd further enhances the affinity of the Phd2−Doc−Phd2 complex for its operator. (C) relBE. Here the mechanism is different and does not involve a low to high affinity switch. Again there are two binding sites for a RelB dimer on the operator. Likely the first binding of a monomeric RelE to both dimeric RelBs on the operator increases the affinity of RelB for the operator via stabilization of its fold through the interactions of RelE with the DNA-binding domain of RelB (see also Figure 2). Addition of a second RelE molecule to one or both operator-bound RelB dimers sterically prevents both RelB dimers from occupying the same operator, leading to release and thus opening the way for transcription. (D) mqsRA. With only a single binding site for MqsA on the mqsRA operator, no conditional cooperativity is observed. Rather, MqsA−MqsR complexes are excluded from operator binding due to steric repulsion between MqsR and the DNA.

their substrates.83−86 Despite that the structural similarities between MazF and CcdB toxins also extend to equivalent antitoxin-binding modes (with identically positioned binding sites), a mechanistic similarity for the regulation of toxin activity seems unlikely (Figure 5C). MazF dimers binds only a single RNA molecule per catalytic cycle.83,85,86 Consequently, the first MazE binds to monomer with the empty RNA binding site. Only binding and partial folding of a second MazE likely pushes the RNA out of its binding site.

[loop S1−S2 (residues Asp16−His28) and loop S3−S4 (residues Thr53−Phe60) in E. coli MazF].78−80 The S1−S2 loop also changes conformation in CcdB, going from gyrasebound to CcdA-bound.40,82 A complex rejuvenation mechanism, on the other hand, is not a priori required for mazEF function. MazE and the RNA substrate may compete in a simple manner for overlapping binding sites on MazF. The crystal structures of the B. subtilis MazF homologue YdcE in complex with a nine-mer RNA substrate analogue and with its MazE-like antitoxin nevertheless show that the N-terminal α-helix of the MazE neutralization domain (Thr50−Ile71) overlaps with the binding groove of the RNA substrate.78 The C-terminal MazE segment, Ser72− Glu82, deviates its path from the RNA binding groove. Hence, antitoxin binding is not in direct competition with a large part of the substrate for binding (Figure 6A). Whether there is an allosteric effect between binding of the substrate and binding of the Ser72−Glu82 as observed for the ccdAB module (see above) is currently unknown, but it is worth mentioning that the catalytic site of MazF itself is not covered in the complex with MazE. MazF proteins are poor RNases with a slow rate of hydrolysis and an unusually high affinity (nanomolar range) for

5.4. Segmental Binding as a General Motif in Antitoxins

The previous examples have in common a segmental binding of the antitoxin to the toxin. The toxin-neutralizing segment consists of two (sometimes three) segments of secondary structure (two pieces of α-helix for CcdA and MazE and an αhelix and a β-strand for RelE) that likely bind consecutively to the toxin starting with the C-terminal segment (summarized in Table 2 for all antitoxins mentioned in this review). Examination of the available structures of toxin−antitoxin complexes shows that this architecture is ubiquitous and not found only in ccdAB, mazEF, and relBE members but also in phd/doc and members of the vapBC family.38,43,56,87−89 In contrast to ccdAB and mazEF, in the phd/doc family, it is the C6942

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the ratio toxin:antitoxin increases, for example, because of proteolytic clearing of the antitoxin, a low-affinity interaction can be replaced by a high-affinity interaction, leading to smaller, soluble complexes of the TAT type, which are poor repressors for simple steric reasons: two TAT complexes cannot bind next to each other on adjacent operator sites (Figure 7A). In this state, the toxin remains neutralized, but transcription is activated, allowing the toxin:antitoxin ratio to recover before the toxin is activated. The vapBC and hipBA families are the only TA modules for which crystal structures of complete toxin−antitoxin−operator complexes are available. vapBC modules, including the related f itAB module from N. gonorrheae, use a variation on the aforementioned architecture. Rather than a linear array of alternating toxin and antitoxin molecules, a closed circular complex is formed consisting of two toxin dimers and two antitoxin dimers. This architecture is observed both in VapBC from Rickettsia felis and FitAB from N. gonorrheae, which differ in the nature of the DNA-binding domain of the antitoxin.43,89 Excess of VapC toxin can disrupt the VapBC operator complex of Salmonalla enterica, which is predicted to be very similar to the R. felis VapBC complex.107 However, the phenomenon is only significant from VapC:VapB ratios of 10 and upward and needs a 20-fold excess of VapC to be complete. Therefore, although it seems that a latent potential for conditional cooperativity is present, its in vivo relevance remains to be validated. The hipBA family is likely an outlier, with the repressor complex consisting of four TA complexes bound to four independent operator sites and unlikely to be regulated through conditional cooperativity.112,113

terminal segment of the neutralization domain that covers part of the ATP binding site of Doc (Figure 6C).17,38 The same is likely also true for VapC proteins, as the C-termini of VapB antitoxins cover or come close to the proposed active sites of VapC proteins.43,87−89 As there is at the moment no structure of a VapC−substrate complex available, steric repulsion between the C-termini of VapB and the substrates of VapCs nevertheless remains speculative. Doc is a member of the larger superfamily of FIC proteins, which catalyze a number of transfer reactions, such as AMPylation, UMPylation, phosphocholination, or phosphorylation of a target protein.90−95 The inhibitory α-helical segment of Phd is here either incorporated into the FIC domain itself as an N- or C-terminal additional helix or again, like in the vbhTA TA-like module from Bartonella schoenbuchensis, as part of a partner protein that is most likely intrinsically disordered in its unbound state.39,96 Although not divided into two distinct segments, the inhibitory helix in all AMPylating FIC members prevents productive AMP binding though steric hindrance by a conserved glutamate residue near the C-terminus of the inhibiting helix.39,97

6. TRANSCRIPTION REGULATION Classic type II TA operons are autoregulated with the antitoxin containing a DNA-binding motif and the toxin modulating the antitoxin-operator affinity.61,98−102 The operator sites vary greatly and usually consist of two or more palindromic or nearpalindromic antitoxin-binding sites,100,103−107 sometimes extended with an additional region that is protected by the bound toxin−antitoxin complex.103,106,108 The geometry of the operator is hereby crucial for functional repression. For example, variations in the relative positions of the two palindromes on the DNA helix abrogates cooperative interactions in the yoeB/yef M module.109

6.2. Low to High Affinity Switch

A switch between a low- and a high-affinity binding mode is believed to be central in the regulation of both ccdAB and phd/ doc.40,56 In both cases, the toxin contains two binding sites for the antitoxin, which differ by several orders of magnitude in affinity. They are further distinguished by different amounts of structuring of the antitoxin upon binding to the toxin. In the repressing complex, both binding sites are occupied. When an excess of toxin is present, additional high-affinity binding sites are presented and the low-affinity interaction can be replaced by a high-affinity interaction, leading to the formation of a nonrepressing TA complex (Figure 7A,B). The molecular details as to how the two affinities are generated differ between ccdAB and phd/doc. CcdB is a homodimer with one antitoxin-binding site on each subunit. However, both binding sites partially overlap, allowing one CcdA C-terminal domain to fully fold upon binding, while the second domain will retain a segment that remains unbound and unfolded (Figure 7A). This results in an extreme case of negative cooperativity and two binding constants that differ by 6 orders of magnitude. Doc, on the other hand, is a monomer and contains two distinct non-overlapping binding sites for the C-terminal region of Phd, differing in hydrophobicity, in the size of the contact surface, and in the conformation that the Phd segment needs to adopt upon binding (Figure 7B). On the basis of structural similarities between CcdB and MazF toxins,40,80 the mechanism in the mazEF family is probably similar to that proposed for ccdAB. In the case of relBE, there seems to be only a single antitoxin-binding site on the toxin RelE, indicating that a high to low affinity switch is

6.1. Conditional Cooperativity

A recurring theme in transcription regulation of type II TA operons is a complex mechanism that has been termed “conditional cooperativity”. This mechanism allows for the toxin to act as a corepressor at low toxin:antitoxin ratios and to become a derepressor at high toxin:antitoxin ratios. Conditional cooperativity has been observed experimentally in five unrelated families of TA modules: ccdAB, relBE, vapBC, mazEF, and phd/doc.27,56,102,107,110 The molecular mechanisms behind conditional cooperativity have been obscure but were recently uncovered for the ccdAB, phd/doc, and relBE families.40,56,111 Three distinct mechanisms can confer conditional cooperativity: (i) a low to high affinity switch in the binding of toxin to antitoxin, (ii) allosteric communication between the DNA-binding and toxin-binding domains of the antitoxin and (iii) steric exclusion between nonrepressing toxin−antitoxin complexes. In the first two mechanisms, intrinsic disorder and protein dynamics contribute directly, while in the third mechanism, the contribution of intrinsic disorder to conditional cooperativity is more indirect. For ccdAB, mazEF, and phd/doc (but not relBE or vapBC), at the toxin:antitoxin ratio corresponding to the highest DNAbinding affinity (and thus tightest repression), a linear ...TATATAT... array of alternating toxin and antitoxin molecules is formed which involves both the high- and the low-affinity binding site on the toxin.27,40,56 Thus, the increase in affinity of the antitoxin for its operator DNA stems from an avidity effect due to the bridging of antitoxins by toxins. When 6943

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RelB, folding of the toxin-binding domain upon binding its target is not likely to directly propagate to the DNA-binding domain of the antitoxin because both domains are connected by a loop region rather than an α-helix. However, folding and binding of the IDP region of the antitoxin to the toxin in these cases results in direct contacts between the toxin and the DNAbinding domain of the antitoxin (Figure 2B,C).80,111 These contacts may stabilize the folded conformation of the DNAbinding domain, thus indirectly affecting DNA binding.

not a contributing factor in the experimentally observed conditional cooperativity.114 The case of vapBC seems to be a variation on the theme where a circular rather than linear AT array consisting of two VapB dimers and two VapC dimers is bound to the operator.43,18 Conditional cooperativity was observed for the vapBC module of Salmonella enterica serovar Typhimurium LT2, but only at very high (>10:1) toxin:antitoxin ratios.107 The crystal structures of the Shigella flexneri VapBC complex and the N. gonorrheae FitAB−operator complex offer an explanation, as neither antitoxin-binding sites on the toxin dimers seem to overlap.43 Thus, competition involves two binding sites with identical or near identical affinities, requiring a large excess of toxin to observe any effect. In contrast, the crystal structure of the R. felis VapBC operator complex again shows partially overlapping binding sites,89 indicating the potential presence of an “affinity switch” mechanism.

6.4. Steric Exclusion

The structures of nonrepressing toxin:antitoxin complexes are highly rigid. In most cases, this rigidity can be attributed to direct interactions between the toxin and the DNA-binding domain of the antitoxin, in addition to the embrace of the neutralizing domain of the antitoxin around the toxin. This is the case for the E. coli MazF2−MazE2−MazF2 and RelE− RelB2−RelE complexes (Figure 2B,C).80,111 In other cases, such as the phd/doc and yoeB/yef M modules, such direct interactions are not present. Yet, as was demonstrated by SAXS measurement, the nonrepressing Doc−Phd2−Doc complex is still a highly rigid entity.56 This rigidity precludes two such complexes to bind simultaneously to adjacent sites on the TA operator (Figure 7A−C). Rigid nonrepressing complexes as such can again on their own be sufficient to generate conditional cooperativity. In such a case, the different antitoxin-binding sites on the operator are located such that repressing complexes (TA 2 or T 2 A 2 architecture) can bind next to each other. The larger nonrepressing complex would not fit because of steric overlap. The driving force for dissociation of the antitoxin from the operator is reduced to the difference in free energy of binding between the antitoxin and operator sites (possibly increased by a single bound toxin through allosteric coupling) and the free energy of the binding of (a second) toxin molecule to the antitoxin. In the “steric exclusion” model, no array of ...TATATAT... links is formed, and therefore, avidity effects will therefore not contribute to the enhanced affinity of the repressor complex for the operator compared to the free antitoxin. The steric exclusion model is the most likely candidate for the mechanism behind conditional cooperativity of the relBE modules (Figure 7C).111 RelE has only a single binding site for RelB and does not dimerize, making the formation of a linear ...TATATAT... array impossible. Modeling of the RelBE operator complex indicates that two RelB dimers can position next to each other, however, without space to position a bridging RelE molecule. A RelE molecule on each side can nevertheless flank a pair of RelB dimers, enhancing the affinity of RelB for its operator sites. Because of the lack of bridging, the RelE-driven increase of affinity of RelB for its operator DNA necessarily has to come from allosteric interactions between both proteins. Likely, binding of a single RelE to a RelB dimer stabilizes the DNAbinding conformation of the ribbon−helix−helix domain of RelB through contacts with RelE,111 increasing its affinity for the operator DNA.110,114

6.3. Allosteric Communication Involving an Intrinsically Disordered Domain

In general, antitoxins are DNA-binding proteins, whose affinity for their operator is increased by their cognate toxin. Thus, the affinity of Phd for a single site operator segment is increased 5− 10-fold in the presence of Doc.56 As the Doc and operator binding sites on Phd are spatially separated and a single site does not allow avidity through bridging of two Phd dimers by one Doc, an allosteric link between the DNA- and Doc-binding sites on Phd needs to be present. Consistent with this notion, the crystal structure of Phd reveals two structural states (Figure 2A).56 In one state, the Nterminal DNA-binding domain is well-folded while the Cterminal 22 amino acid Doc-binding segment remains disordered. The region connecting both the DNA-binding domain and the C-terminal disordered domain adopts an αhelical conformation. In its other state, the DNA-binding domain contains significant local disorder as well. Its central βsheet is retained, but the surrounding α-helices, including the αhelix linking to the Doc-binding segment, partially or completely unwind. As a result, the DNA-binding site is disturbed and DNA binding will result in a significant entropic penalty. The allosteric communication between the Doc- and DNAbinding sites on Phd can be explained in terms of stabilization of the connecting α-helix between both Phd domains.56 Binding of Doc to residues Leu52−Arg73 of Phd induces an α-helix that extends into the connecting helix Lys41−Leu52. The stabilization of the latter then propagates to the DNAbinding domain, resulting in tighter DNA binding. Further evidence for this mechanism comes from mutations in the region linking both domains that uncouple toxin neutralization from transcription regulation. These mutants bind and inhibit Doc in a wild-type manner yet fail to increase their affinity for DNA in the presence of Doc.56 Allosteric communication between the two antitoxin domains has not been investigated for antitoxins other than Phd. Given its close structural similarity with Phd and the conservation of some key features that are believed to be important for the interdomain communication, YefM is expected to act in a similar way as Phd.42 The architecture of the C. crescentus ParDE complex is also compatible with a similar mechanism.41 Other antitoxins may exhibit similar properties but based upon other molecular principles. For example, in MazE and

6.5. A Simplified Version of Conditional Cooperativity in TA Modules with a Single Operator Site

Conditional cooperativity in TA regulation was defined as a dose effect of the toxin on the DNA-binding properties of the antitoxin. While different molecular mechanisms can be at play, one constant seems to be that at least two closely spaced 6944

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Biographies

antitoxin-binding sites need to be present on the operator DNA. There are, nevertheless, TA modules where the operator contains only a single palindrome that can be recognized by the antitoxin dimer. In theory, toxin-induced interdomain allosteric communication might be enough to induce conditional cooperativity. All that is needed for this is that binding of one or two toxins to the antitoxin dimer has differential effects. This could lead to a situation where the affinity for the operator DNA of the antitoxin dimer with a single toxin bound is tighter than the affinities of the free antitoxin dimer as well as of the antitoxin dimer with two toxin species bound. This mechanism would allow conditional cooperativity to occur if there is only a single binding site on the operator. Multiple antitoxin-binding sites on the operator would increase the effect of conditional cooperativity, even if the bound toxin−antitoxin complexes do not physically touch each other, a situation found in the hipAB operator.112,113 Conditional cooperativity using single site operators has until now not been demonstrated, and the aforementioned mechanism, although plausible, remains speculative. The E. coli mqsRA module is the sole TA module where a mechanism was proposed for an embryonic form of conditional cooperativity that is able to act at the level of single operator sites (although the full operator still contains two binding sites for MqsA). It was shown that MqsR does not increase the affinity of MqsA for its operator sequence, even at low MqsR:MqsA ratios.115 Rather, MqsR acts solely as a transcriptional derepressor and destabilizes MqsA:operator complexes via steric hindrance between MqsR and the operator DNA (Figure 7D). In this way, the mqsRA module is still able to counteract the generation of an excess of toxin but using a mechanism that is more simple than full conditional cooperativity.

Remy Loris received his Ph.D. at the Vrije Universiteit Brussel (VUB) in 1994 on the structural biology of plant lectins. Apart from two short postdocs in Leicester and Grenoble, he stayed at his alma mater, where he became research professor in 2003. In 2011, he also became group leader at the Flanders Institute for Biotechnology (VIB). He was trained initially as a protein crystallographer, but now uses a variety of structural biology and biophysics methods to study prokaryotic toxin− antitoxin modules. Using these small protein networks as model systems, he attempts to answer questions related to molecular recognition and intramolecular signaling in proteins, with a specific interest in mechanisms based on dynamics and intrinsic disorder.

7. CONCLUSIONS In summary, bacterial toxin−antitoxin modules use intrinsically disordered segments for regulation at the level of protein activity and transcription through a series of parallel and conceptually similar mechanisms, but differing in molecular details. They exemplify the variety of functionalities that can arise from simple folding-upon-binding interactions. Intrinsic disorder and protein flexibility in general are crucial for the reversibility of TA action, allowing, for example, rejuvenation of CcdB-poisoned gyrase. It is equally crucial for keeping the toxin:antitoxin ratio constant and thus avoiding accidental toxin activation. The latter involves a regulatory mechanism termed “conditional cooperativity” that can employ intrinsic disorder and structural flexibility via at least two different molecular mechanisms. Increased operator affinity of the toxin−antitoxin complex compared to the antitoxin results from either bridging pairs of antitoxins by the toxin (with the consequent avidity effect) or from an allosteric effect of the toxin on the antitoxin. Disruption of the operator complex at higher toxin to antitoxin ratios results from steric hindrance between two neighboring toxins or between toxin and operator DNA.

Abel Garcia-Pino received his B.S. at the University of Havana, Cuba, and completed his Ph.D. in 2010 at the Vrije Universiteit Brussel (VUB), after completing an M.S. in Molecular Biology. He is presently an FWO-funded postdoctoral fellow at VUB. As a biochemist his research career focuses on understanding biological processes at the molecular level. His current research centers on the role of intrinsically disordered proteins (IDPs) in transcription regulation and the structure and biophysical aspects of allosteric signal transduction involving IDPs.

ACKNOWLEDGMENTS We thank Kenn Gerdes for critical reading of the manuscript. This work was possible through grants from FWO-Vlaanderen, VIB, and OZR-VUB. The authors are indebted to Lieven Buts for help with the figures. A.G.-P. is an FWO postdoctoral fellow.

AUTHOR INFORMATION Corresponding Author

REFERENCES

*E-mail: [email protected].

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Notes

The authors declare no competing financial interest. 6945

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