Natalie De Jonge

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.

Driving Forces of Gyrase Recognition by the Addiction Toxin CcdB

Gyrase, an essential bacterial topoisomerase, is the target of several antibiotics (e.g. quinolones) as well as of bacterial toxin CcdB. This toxin, encoded by Escherichia coli toxin-antitoxin module ccd, poisons gyrase by causing inhibition of both transcription and replication. Because the molecular driving forces of gyrase unfolding and CcdB-gyrase binding were unknown, the nature of the CcdB-gyrase recognition remained elusive. Therefore, we performed a detailed thermodynamic analysis of CcdB binding to several fragments of gyrase A subunit (GyrA) that contain the CcdB-binding site. Binding of CcdB to the shorter fragments was studied directly by isothermal titration calorimetry. Its binding to the longer GyrA59 fragment in solution is kinetically limited and was therefore investigated via urea induced unfolding of the GyrA59-CcdB complex and unbound GyrA59 and CcdB, monitored by circular dichroism spectroscopy. Model analysis of experimental data, in combination with the relevant structural information, indicates that CcdB binding to gyrase is an enthalpic process driven mainly by specific interactions between CcdB and the highly stable dimerization domain of the GyrA. The dissection of binding energetics indicates that CcdB-gyrase recognition is accompanied by opening of the tower and catalytic domain of GyrA. Such extensive structural rearrangements appear to be crucial driving forces for the functioning of the ccd toxin-antitoxin module.

Structural and Thermodynamic Characterization of Vibrio fischeri CcdB

CcdBVfi from Vibrio fischeri is a member of the CcdB family of toxins that poison covalent gyrase-DNA complexes. In solution CcdBVfi 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, CcdBVfi possesses a rigid dimer interface, and the apparent relative rotations of the two subunits are due to structural plasticity of the monomer. CcdBVfi 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.

Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF.

Escherichia coli MazF (EcMazF) is the archetype of a large family of ribonucleases involved in bacterial stress response. The crystal structure of EcMazF in complex with a 7-nucleotide substrate mimic explains the relaxed substrate specificity of the E. coli enzyme relative to its Bacillus subtilis counterpart and provides a framework for rationalizing specificity in this enzyme family. In contrast to a conserved mode of substrate recognition and a conserved active site, regulation of enzymatic activity by the antitoxin EcMazE diverges from its B. subtilis homolog. Central in this regulation is an EcMazE-induced double conformational change as follows: a rearrangement of a crucial active site loop and a relative rotation of the two monomers in the EcMazF dimer. Both are induced by the C-terminal residues Asp-78–Trp-82 of EcMazE, which are also responsible for strong negative cooperativity in EcMazE-EcMazF binding. This situation shows unexpected parallels to the regulation of the F-plasmid CcdB activity by CcdA and further supports a common ancestor despite the different activities of the MazF and CcdB toxins. In addition, we pinpoint the origin of the lack of activity of the E24A point mutant of EcMazF in its inability to support the substrate binding-competent conformation of EcMazF.

Alternative interactions define gyrase specificity in the CcdB family.

Toxin–antitoxin (TA) modules are small operons associated with stress response of bacteria. F‐plasmid CcdBF 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 CcdBVfi 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.

Purification and crystallization of Vibrio fischeri CcdB and its complexes with fragments of gyrase and CcdA.

The ccd toxin-antitoxin module from the Escherichia coli F plasmid has a homologue on the Vibrio fischeri integron. The homologue of the toxin (CcdB(Vfi)) was crystallized in two different crystal forms. The first form belongs to space group I23 or I2(1)3, with unit-cell parameter a = 84.5 A, and diffracts to 1.5 A resolution. The second crystal form belongs to space group C2, with unit-cell parameters a = 58.5, b = 43.6, c = 37.5 A, beta = 110.0 degrees, and diffracts to 1.7 A resolution. The complex of CcdB(Vfi) with the GyrA14(Vfi) fragment of V. fischeri gyrase crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 53.5, b = 94.6, c = 58.1 A, and diffracts to 2.2 A resolution. The corresponding mixed complex with E. coli GyrA14(Ec) crystallizes in space group C2, with unit-cell parameters a = 130.1, b = 90.8, c = 58.1 A, beta = 102.6 degrees, and diffracts to 1.95 A. Finally, a complex between CcdB(Vfi) and part of the F-plasmid antitoxin CcdA(F) crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 46.9, b = 62.6, c = 82.0 A, and diffracts to 1.9 A resolution.

Crystallization of the C-terminal domain of the addiction antidote CcdA in complex with its toxin CcdB

CcdA and CcdB are the antidote and toxin of the ccd addiction module of Escherichia coli plasmid F. The CcdA C-terminal domain (CcdAC36; 36 amino acids) was crystallized in complex with CcdB (dimer of 2 x 101 amino acids) in three different crystal forms, two of which diffract to high resolution. Form II belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 37.6, b = 60.5, c = 83.8 A and diffracts to 1.8 A resolution. Form III belongs to space group P2(1), with unit-cell parameters a = 41.0, b = 37.9, c = 69.6 A, beta = 96.9 degrees, and diffracts to 1.9 A resolution.

Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies

Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe229 and Phe279 of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe279. Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe279. In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe279, whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe229.

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 1H, 15N and 13C resonance assignments of CcdBVfi from the marine bacterium Vibrio fischeri. The BMRB accession number is 16135.