Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae.

Disordered protein sequences can exhibit different binding modes, ranging from well-ordered folding-upon-binding to highly dynamic fuzzy binding. The primary function of the intrinsically disordered region of the antitoxin HigA2 from Vibrio cholerae is to neutralize HigB2 toxin through ultra-high-affinity folding-upon-binding interaction. Here, we show that the same intrinsically disordered region can also mediate fuzzy interactions with its operator DNA and, through interplay with the folded helix-turn-helix domain, regulates transcription from the higBA2 operon. NMR, SAXS, ITC and in vivo experiments converge towards a consistent picture where a specific set of residues in the intrinsically disordered region mediate electrostatic and hydrophobic interactions while "hovering" over the DNA operator. Sensitivity of the intrinsically disordered region to scrambling the sequence, position-specific contacts and absence of redundant, multivalent interactions, point towards a more specific type of fuzzy binding. Our work demonstrates how a bacterial regulator achieves dual functionality by utilizing two distinct interaction modes within the same disordered sequence.

Ribosome-dependent Vibrio cholerae mRNAse HigB2 is regulated by a ß-strand sliding mechanism.

Toxin-antitoxin (TA) modules are small operons involved in bacterial stress response and persistence. higBA operons form a family of TA modules with an inverted gene organization and a toxin belonging to the RelE/ParE superfamily. Here, we present the crystal structures of chromosomally encoded Vibrio cholerae antitoxin (VcHigA2), toxin (VcHigB2) and their complex, which show significant differences in structure and mechanisms of function compared to the higBA module from plasmid Rts1, the defining member of the family. The VcHigB2 is more closely related to Escherichia coli RelE both in terms of overall structure and the organization of its active site. VcHigB2 is neutralized by VcHigA2, a modular protein with an N-terminal intrinsically disordered toxin-neutralizing segment followed by a C-terminal helix-turn-helix dimerization and DNA binding domain. VcHigA2 binds VcHigB2 with picomolar affinity, which is mainly a consequence of entropically favorable de-solvation of a large hydrophobic binding interface and enthalpically favorable folding of the N-terminal domain into an α-helix followed by a ß-strand. This interaction displaces helix α3 of VcHigB2 and at the same time induces a one-residue shift in the register of ß-strand ß3, thereby flipping the catalytically important Arg64 out of the active site.

Crystallization and preliminary X-ray analysis of four cysteine proteases from Ficus carica latex.

The latex of the common fig (Ficus carica) contains a mixture of at least five cysteine proteases commonly known as ficins (EC 3.4.22.3). Four of these proteases were purified to homogeneity and crystals were obtained in a variety of conditions. The four ficin (iso)forms appear in ten different crystal forms. All diffracted to better than 2.10 Šresolution and for each form at least one crystal form diffracted to 1.60 Šresolution or higher. Ficin (iso)forms B and C share a common crystal form, suggesting close sequence and structural similarity. The latter diffracted to a resolution of 1.20 Šand belonged to space group P3121 or P3221, with unit-cell parameters a = b = 88.9, c = 55.9 Å.

An efficient method for the purification of proteins from four distinct toxin-antitoxin modules.

Toxin-antitoxin (TA) modules are stress response elements that are ubiquitous in the genomes of bacteria and archaea. Production and subsequent purification of individual TA proteins is anything but straightforward as over-expression of the toxin gene is lethal to bacterial and eukaryotic cells and over-production of the antitoxin leads to its proteolytic degradation because of its inherently unstructured nature. Here we describe an effective production and purification strategy centered on an on-column denaturant-induced dissociation of the toxin-antitoxin complex. The success of the method is demonstrated by its application on four different TA families, encoding proteins with distinct activities and folds. A series of biophysical and in vitro activity tests show that the purified proteins are of high quality and suitable for structural studies.

The intrinsically disordered domain of the antitoxin Phd chaperones the toxin Doc against irreversible inactivation and misfolding.

The toxin Doc from the phd/doc toxin-antitoxin module targets the cellular translation machinery and is inhibited by its antitoxin partner Phd. Here we show that Phd also functions as a chaperone, keeping Doc in an active, correctly folded conformation. In the absence of Phd, Doc exists in a relatively expanded state that is prone to dimerization through domain swapping with its active site loop acting as hinge region. The domain-swapped dimer is not capable of arresting protein synthesis in vitro, whereas the Doc monomer is. Upon binding to Phd, Doc becomes more compact and is secured in its monomeric state with a neutralized active site.

Crystallization and preliminary X-ray analysis of two variants of the Escherichia coli O157 ParE2-PaaA2 toxin-antitoxin complex.

The paaR2-paaA2-parE2 operon is a three-component toxin-antitoxin module encoded in the genome of the human pathogen Escherichia coli O157. The toxin (ParE2) and antitoxin (PaaA2) interact to form a nontoxic toxin-antitoxin complex. In this paper, the crystallization and preliminary characterization of two variants of the ParE2-PaaA2 toxin-antitoxin complex are described. Selenomethionine-derivative crystals of the full-length ParE2-PaaA2 toxin-antitoxin complex diffracted to 2.8 Šresolution and belonged to space group P41212 (or P43212), with unit-cell parameters a = b = 90.5, c = 412.3 Å. It was previously reported that the full-length ParE2-PaaA2 toxin-antitoxin complex forms a higher-order oligomer. In contrast, ParE2 and PaaA213-63, a truncated form of PaaA2 in which the first 12 N-terminal residues of the antitoxin have been deleted, form a heterodimer as shown by analytical gel filtration, dynamic light scattering and small-angle X-ray scattering. Crystals of the PaaA213-63-ParE2 complex diffracted to 2.7 Šresolution and belonged to space group P6122 (or P6522), with unit-cell parameters a = b = 91.6, c = 185.6 Å.

Structural and biophysical characterization of Staphylococcus aureus SaMazF shows conservation of functional dynamics.

The Staphylococcus aureus genome contains three toxin-antitoxin modules, including one mazEF module, SamazEF. Using an on-column separation protocol we are able to obtain large amounts of wild-type SaMazF toxin. The protein is well-folded and highly resistant against thermal unfolding but aggregates at elevated temperatures. Crystallographic and nuclear magnetic resonance (NMR) solution studies show a well-defined dimer. Differences in structure and dynamics between the X-ray and NMR structural ensembles are found in three loop regions, two of which undergo motions that are of functional relevance. The same segments also show functionally relevant dynamics in the distantly related CcdB family despite divergence of function. NMR chemical shift mapping and analysis of residue conservation in the MazF family suggests a conserved mode for the inhibition of MazF by MazE.

Energetic basis of uncoupling folding from binding for an intrinsically disordered protein.

Intrinsically disordered proteins (IDPs) are proteins that lack a unique three-dimensional structure in their native state. Many have, however, been found to fold into a defined structure when interacting with specific binding partners. The energetic implications of such behavior have been widely discussed, yet experimental thermodynamic data is scarce. We present here a thorough thermodynamic and structural study of the binding of an IDP (antitoxin CcdA) to its molecular target (gyrase poison CcdB). We show that the binding-coupled folding of CcdA is driven by a combination of specific intramolecular interactions that favor the final folded structure and a less specific set of intermolecular contacts that provide a desolvation entropy boost. The folded structure of the bound IDP appears to be defined largely by its own amino acid sequence, with the binding partner functioning more as a facilitator than a mold to conform to. On the other hand, specific intermolecular interactions do increase the binding affinity up to the picomolar range. Overall, this study shows how an IDP can achieve very strong and structurally well-defined binding and it provides significant insight into the molecular forces that enable such binding properties.

The ParE2-PaaA2 toxin-antitoxin complex from Escherichia coli O157 forms a heterodocecamer in solution and in the crystal.

Escherichia coli O157 paaR2-paaA2-parE2 constitutes a unique three-component toxin-antitoxin (TA) module encoding a toxin (ParE2) related to the classic parDE family but with an unrelated antitoxin called PaaA2. The complex between PaaA2 and ParE2 was purified and characterized by analytical gel filtration, dynamic light scattering and small-angle X-ray scattering. It consists of a particle with a radius of gyration of 3.95 nm and is likely to form a heterododecamer. Crystals of the ParE2-PaaA2 complex diffract to 3.8 Å resolution and belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 142.9, c = 87.5 Å. The asymmetric unit is consistent with a particle of around 125 kDa, which is compatible with the solution data. Therefore, the ParE2-PaaA2 complex is the largest toxin-antitoxin complex identified to date and its quaternary arrangement is likely to be of biological significance.

Alternative interactions define gyrase specificity in the CcdB family.

Toxin-antitoxin (TA) modules are small operons associated with stress response of bacteria. F-plasmid CcdB(F) was the first TA toxin for which its target, gyrase, was identified. Plasmidic and chromosomal CcdBs belong to distinct families. Conserved residues crucial for gyrase poisoning activity of plasmidic CcdBs are not conserved among these families. Here we show that the chromosomal CcdB(Vfi) from Vibrio fischeri is an active gyrase poison that interacts with its target via an alternative energetic mechanism. Changes in the GyrA14-binding surface of the Vibrio and F-plasmid CcdB family members illustrate neutral drift where alternative interactions can be used to achieve the same functionality. Differences in affinity between V. fischeri and F-plasmid CcdB for gyrase and their corresponding CcdA antitoxin possibly reflect distinct roles for TA modules located on plasmids and chromosomes.

Crystallization of the Staphylococcus aureus MazF mRNA interferase.

mazEF modules encode toxin-antitoxin pairs that are involved in the bacterial stress response through controlled and specific degradation of mRNA. Staphylococcus aureus MazF and MazE constitute a unique toxin-antitoxin module under regulation of the sigB operon. A MazF-type mRNA interferase is combined with an antitoxin of unknown fold. Crystals of S. aureus MazF (SaMazF) were grown in space group P2(1)2(1)2(1). The crystals diffracted to 2.1 Šresolution and are likely to contain two SaMazF dimers in the asymmetric unit.

1H, 13C, and 15N backbone and side-chain chemical shift assignment of the staphylococcal MazF mRNA interferase.

MazF proteins are ribonucleases that cleave mRNA with high sequence-specificity as part of bacterial stress response and that are neutralized by the action of the corresponding antitoxin MazE. Prolonged activation of the toxin MazF leads to cell death. Several mazEF modules from gram-negative bacteria have been characterized in terms of catalytic activity, auto-regulation mechanism and structure, but less is known about their distant relatives found in gram-positive organisms. Currently, no solution NMR structure is available for any wild-type MazF toxin. Here we report the (1)H, (15)N and (13)C backbone and side-chain chemical shift assignments of this toxin from the pathogen bacterium Staphylococcus aureus. The BMRB accession number is 17288.

Structural and thermodynamic characterization of Vibrio fischeri CcdB.

CcdB(Vfi) from Vibrio fischeri is a member of the CcdB family of toxins that poison covalent gyrase-DNA complexes. In solution CcdB(Vfi) is a dimer that unfolds to the corresponding monomeric components in a two-state fashion. In the unfolded state, the monomer retains a partial secondary structure. This observation correlates well with the crystal and NMR structures of the protein, which show a dimer with a hydrophobic core crossing the dimer interface. In contrast to its F plasmid homologue, CcdB(Vfi) possesses a rigid dimer interface, and the apparent relative rotations of the two subunits are due to structural plasticity of the monomer. CcdB(Vfi) shows a number of non-conservative substitutions compared with the F plasmid protein in both the CcdA and the gyrase binding sites. Although variation in the CcdA interaction site likely determines toxin-antitoxin specificity, substitutions in the gyrase-interacting region may have more profound functional implications.

Rejuvenation of CcdB-poisoned gyrase by an intrinsically disordered protein domain.

Toxin-antitoxin modules are small regulatory circuits that ensure survival of bacterial populations under challenging environmental conditions. The ccd toxin-antitoxin module on the F plasmid codes for the toxin CcdB and its antitoxin CcdA. CcdB poisons gyrase while CcdA actively dissociates CcdB:gyrase complexes in a process called rejuvenation. The CcdA:CcdB ratio modulates autorepression of the ccd operon. The mechanisms behind both rejuvenation and regulation of expression are poorly understood. We show that CcdA binds consecutively to two partially overlapping sites on CcdB, which differ in affinity by six orders of magnitude. The first, picomolar affinity interaction triggers a conformational change in CcdB that initiates the dissociation of CcdB:gyrase complexes by an allosteric segmental binding mechanism. The second, micromolar affinity binding event regulates expression of the ccd operon. Both functions of CcdA, rejuvenation and autoregulation, are mechanistically intertwined and depend crucially on the intrinsically disordered nature of the CcdA C-terminal domain.

Sequence-specific 1H, 15N and 13C resonance assignments of the 23.7-kDa homodimeric toxin CcdB from Vibrio fischeri.

CcdB is the toxic component of a bacterial toxin-antitoxin system. It inhibits DNA gyrase (a type II topoisomerase), and its toxicity can be neutralized by binding of its antitoxin CcdA. Here we report the sequential backbone and sidechain (1)H, (15)N and (13)C resonance assignments of CcdB(Vfi) from the marine bacterium Vibrio fischeri. The BMRB accession number is 16135.