Allostery and intrinsic disorder mediate transcription regulation by conditional cooperativity.

Regulation of the phd/doc toxin-antitoxin operon involves the toxin Doc as co- or derepressor depending on the ratio between Phd and Doc, a phenomenon known as conditional cooperativity. The mechanism underlying this observed behavior is not understood. Here we show that monomeric Doc engages two Phd dimers on two unrelated binding sites. The binding of Doc to the intrinsically disordered C-terminal domain of Phd structures its N-terminal DNA-binding domain, illustrating allosteric coupling between highly disordered and highly unstable domains. This allosteric effect also couples Doc neutralization to the conditional regulation of transcription. In this way, higher levels of Doc tighten repression up to a point where the accumulation of toxin triggers the production of Phd to counteract its action. Our experiments provide the basis for understanding the mechanism of conditional cooperative regulation of transcription typical of toxin-antitoxin modules. This model may be applicable for the regulation of other biological systems.

Arabidopsis casein kinase 2 triggers stem cell exhaustion under Al toxicity and phosphate deficiency through activating the DNA damage response pathway.

Aluminum (Al) toxicity and inorganic phosphate (Pi) limitation are widespread chronic abiotic and mutually enhancing stresses that profoundly affect crop yield. Both stresses strongly inhibit root growth, resulting from a progressive exhaustion of the stem cell niche. Here, we report on a casein kinase 2 (CK2) inhibitor identified by its capability to maintain a functional root stem cell niche in Arabidopsis thaliana under Al toxic conditions. CK2 operates through phosphorylation of the cell cycle checkpoint activator SUPPRESSOR OF GAMMA RADIATION1 (SOG1), priming its activity under DNA-damaging conditions. In addition to yielding Al tolerance, CK2 and SOG1 inactivation prevents meristem exhaustion under Pi starvation, revealing the existence of a low Pi-induced cell cycle checkpoint that depends on the DNA damage activator ATAXIA-TELANGIECTASIA MUTATED (ATM). Overall, our data reveal an important physiological role for the plant DNA damage response pathway under agriculturally limiting growth conditions, opening new avenues to cope with Pi limitation.

Evaluation of different strategies to produce Vibrio cholerae ParE2 toxin.

Toxin-antitoxin (TA) systems are small operons that are omnipresent in bacteria and archaea with suggested roles in stabilization of mobile genetic elements, bacteriophage protection, stress response and possibly persister formation. A major bottleneck in the study of TA toxins is the production of sufficient amounts of well-folded, functional protein. Here we examine alternative approaches for obtaining the VcParE2 toxin from Vibrio cholerae. VcParE2 can be successfully produced via bacterial expression in presence of its cognate antitoxin VcParD2, followed by on-column unfolding and refolding. Alternatively, the toxin can be expressed in Spodoptera frugiperda (Sf9) insect cells. The latter requires disruption of the VcparE2 gene via introduction of an insect cell intron. Both methods provide protein with similar structural and functional characteristics.

The sequence-ensemble relationship in fuzzy protein complexes.

Intrinsically disordered proteins (IDPs) interact with globular proteins through a variety of mechanisms, resulting in the structurally heterogeneous ensembles known as fuzzy complexes. While there exists a reasonable comprehension on how IDP sequence determines the unbound IDP ensemble, little is known about what shapes the structural characteristics of IDPs bound to their targets. Using a statistical thermodynamic model, we show that the target-bound ensembles are determined by a simple code that combines the IDP sequence and the distribution of IDP-target interaction hotspots. These two parameters define the conformational space of target-bound IDPs and rationalize the observed structural heterogeneity of fuzzy complexes. The presented model successfully reproduces the dynamical signatures of target-bound IDPs from the NMR relaxation experiments as well as the changes of interaction affinity and the IDP helicity induced by mutations. The model explains how the target-bound IDP ensemble adapts to mutations in order to achieve an optimal balance between conformational freedom and interaction energy. Taken together, the presented sequence-ensemble relationship of fuzzy complexes explains the different manifestations of IDP disorder in folding-upon-binding processes.

A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures.

Coiled-coil (CC) dimers are widely used in protein design because of their modularity and well-understood sequence-structure relationship. In CC protein origami design, a polypeptide chain is assembled from a defined sequence of CC building segments that determine the self-assembly of protein cages into polyhedral shapes, such as the tetrahedron, triangular prism, or four-sided pyramid. However, a targeted functionalization of the CC modules could significantly expand the versatility of protein origami scaffolds. Here, we describe a panel of single-chain camelid antibodies (nanobodies) directed against different CC modules of a de novo designed protein origami tetrahedron. We show that these nanobodies are able to recognize the same CC modules in different polyhedral contexts, such as isolated CC dimers, tetrahedra, triangular prisms, or trigonal bipyramids, thereby extending the ability to functionalize polyhedra with nanobodies in a desired stoichiometry. Crystal structures of five nanobody-CC complexes in combination with small-angle X-ray scattering show binding interactions between nanobodies and CC dimers forming the edges of a tetrahedron with the nanobody entering the tetrahedral cavity. Furthermore, we identified a pair of allosteric nanobodies in which the binding to the distant epitopes on the antiparallel homodimeric APH CC is coupled via a strong positive cooperativity. A toolbox of well-characterized nanobodies specific for CC modules provides a unique tool to target defined sites in the designed protein structures, thus opening numerous opportunities for the functionalization of CC protein origami polyhedra or CC-based bionanomaterials.

Alternative dimerization is required for activity and inhibition of the HEPN ribonuclease RnlA.

The rnlAB toxin-antitoxin operon from Escherichia coli functions as an anti-phage defense system. RnlA was identified as a member of the HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) superfamily of ribonucleases. The activity of the toxin RnlA requires tight regulation by the antitoxin RnlB, the mechanism of which remains unknown. Here we show that RnlA exists in an equilibrium between two different homodimer states: an inactive resting state and an active canonical HEPN dimer. Mutants interfering with the transition between states show that canonical HEPN dimerization via the highly conserved RX4-6H motif is required for activity. The antitoxin RnlB binds the canonical HEPN dimer conformation, inhibiting RnlA by blocking access to its active site. Single-alanine substitutions mutants of the highly conserved R255, E258, R318 and H323 show that these residues are involved in catalysis and substrate binding and locate the catalytic site near the dimer interface of the canonical HEPN dimer rather than in a groove located between the HEPN domain and the preceding TBP-like domain. Overall, these findings elucidate the structural basis of the activity and inhibition of RnlA and highlight the crucial role of conformational heterogeneity in protein function.

Structure and substrate specificity determinants of the taurine biosynthetic enzyme cysteine sulphinic acid decarboxylase.

Pyridoxal 5́-phosphate (PLP) is an important cofactor for amino acid decarboxylases with many biological functions, including the synthesis of signalling molecules, such as serotonin, dopamine, histamine, γ-aminobutyric acid, and taurine. Taurine is an abundant amino acid with multiple physiological functions, including osmoregulation, pH regulation, antioxidative protection, and neuromodulation. In mammalian tissues, taurine is mainly produced by decarboxylation of cysteine sulphinic acid to hypotaurine, catalysed by the PLP-dependent cysteine sulphinic acid decarboxylase (CSAD), followed by oxidation of the product to taurine. We determined the crystal structure of mouse CSAD and compared it to other PLP-dependent decarboxylases in order to identify determinants of substrate specificity and catalytic activity. Recognition of the substrate involves distinct side chains forming the substrate-binding cavity. In addition, the backbone conformation of a buried active-site loop appears to be a critical determinant for substrate side chain binding in PLP-dependent decarboxylases. Phe94 was predicted to affect substrate specificity, and its mutation to serine altered both the catalytic properties of CSAD and its stability. Using small-angle X-ray scattering, we further showed that CSAD presents open/close motions in solution. The structure of apo-CSAD indicates that the active site gets more ordered upon internal aldimine formation. Taken together, the results highlight details of substrate recognition in PLP-dependent decarboxylases and provide starting points for structure-based inhibitor design with the aim of affecting the biosynthesis of taurine and other abundant amino acid metabolites.

Thermodynamic Stability of the Transcription Regulator PaaR2 from Escherichia coli O157:H7.

PaaR2 is a putative transcription regulator encoded by a three-component parDE-like toxin-antitoxin module from Escherichia coli O157:H7. Although this module's toxin, antitoxin, and toxin-antitoxin complex have been more thoroughly investigated, little remains known about its transcription regulator PaaR2. Using a wide range of biophysical techniques (circular dichroism spectroscopy, size-exclusion chromatography-multiangle laser light scattering, dynamic light scattering, small-angle x-ray scattering, and native mass spectrometry), we demonstrate that PaaR2 mainly consists of α-helices and displays a concentration-dependent octameric build-up in solution and that this octamer contains a global shape that is significantly nonspherical. Thermal unfolding of PaaR2 is reversible and displays several transitions, suggesting a complex unfolding mechanism. The unfolding data obtained from spectroscopic and calorimetric methods were combined into a unifying thermodynamic model, which suggests a five-state unfolding trajectory. Furthermore, the model allows the calculation of a stability phase diagram, which shows that, under physiological conditions, PaaR2 mainly exists as a dimer that can swiftly oligomerize into an octamer depending on local protein concentrations. These findings, based on a thorough biophysical and thermodynamic analysis of PaaR2, may provide important insights into biological function such as DNA binding and transcriptional regulation.

The quaternary structure of human tyrosine hydroxylase: effects of dystonia-associated missense variants on oligomeric state and enzyme activity.

Tyrosine hydroxylase (TH) is a multi-domain, homo-oligomeric enzyme that catalyses the rate-limiting step of catecholamine neurotransmitter biosynthesis. Missense variants of human TH are associated with a recessive neurometabolic disease with low levels of brain dopamine and noradrenaline, resulting in a variable clinical picture, from progressive brain encephalopathy to adolescent onset DOPA-responsive dystonia (DRD). We expressed isoform 1 of human TH (hTH1) and its dystonia-associated missense variants in E. coli, analysed their quaternary structure and thermal stability using size-exclusion chromatography, circular dichroism, multi-angle light scattering, transmission electron microscopy, small-angle X-ray scattering and assayed hydroxylase activity. Wild-type (WT) hTH1 was a mixture of enzymatically stable tetramers (85.6%) and octamers (14.4%), with little interconversion between these species. We also observed small amounts of higher order assemblies of long chains of enzyme by transmission electron microscopy. To investigate the role of molecular assemblies in the pathogenesis of DRD, we compared the structure of WT hTH1 with the DRD-associated variants R410P and D467G that are found in vicinity of the predicted subunit interfaces. In contrast to WT hTH1, R410P and D467G were mixtures of tetrameric and dimeric species. Inspection of the available structures revealed that Arg-410 and Asp-467 are important for maintaining the stability and oligomeric structure of TH. Disruption of the normal quaternary enzyme structure by missense variants is a new molecular mechanism that may explain the loss of TH enzymatic activity in DRD. Unstable missense variants could be targets for pharmacological intervention in DRD, aimed to re-establish the normal oligomeric state of TH.

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.

Molecular mechanism governing ratio-dependent transcription regulation in the ccdAB operon.

Bacteria can become transiently tolerant to several classes of antibiotics. This phenomenon known as persistence is regulated by small genetic elements called toxin-antitoxin modules with intricate yet often poorly understood self-regulatory features. Here, we describe the structures of molecular complexes and interactions that drive the transcription regulation of the ccdAB toxin-antitoxin module. Low specificity and affinity of the antitoxin CcdA2 for individual binding sites on the operator are enhanced by the toxin CcdB2, which bridges the CcdA2 dimers. This results in a unique extended repressing complex that spirals around the operator and presents equally spaced DNA binding sites. The multivalency of binding sites induces a digital on-off switch for transcription, regulated by the toxin:antitoxin ratio. The ratio at which this switch occurs is modulated by non-specific interactions with the excess chromosomal DNA. Altogether, we present the molecular mechanisms underlying the ratio-dependent transcriptional regulation of the ccdAB operon.

The many faces of Fic: structural and functional aspects of Fic enzymes.

Fic enzymes post-translationally modify proteins through AMPylation, UMPylation, phosphorylation, or phosphocholination. They have been identified across all domains of life, and they target a myriad of proteins such as eukaryotic GTPases, unstructured protein segments, and bacterial enzymes. Consequently, they play crucial roles in eukaryotic signal transduction, drug tolerance, bacterial pathogenicity, and the bacterial stress response. Structurally, they consist of an all α-helical core domain that supports and scaffolds a structurally conserved active-site loop, which catalyses the transfer of various parts of a nucleotide cofactor to proteins. Despite their diverse substrates and targets, they retain a conserved active site and reaction chemistry. This catalytic variety came to light only recently with the crystal structures of different Fic enzymes.

An intrinsically disordered entropic switch determines allostery in Phd-Doc regulation.

Conditional cooperativity is a common mechanism involved in transcriptional regulation of prokaryotic type II toxin-antitoxin operons and is intricately related to bacterial persistence. It allows the toxin component of a toxin-antitoxin module to act as a co-repressor at low doses of toxin as compared to antitoxin. When toxin level exceeds a certain threshold, however, the toxin becomes a de-repressor. Most antitoxins contain an intrinsically disordered region (IDR) that typically is involved in toxin neutralization and repressor complex formation. To address how the antitoxin IDR is involved in transcription regulation, we studied the phd-doc operon from bacteriophage P1. We provide evidence that the IDR of Phd provides an entropic barrier precluding full operon repression in the absence of Doc. Binding of Doc results in a cooperativity switch and consequent strong operon repression, enabling context-specific modulation of the regulatory process. Variations of this theme are likely to be a common mechanism in the autoregulation of bacterial operons that involve intrinsically disordered regions.

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 Phe(229) and Phe(279) of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe(279) 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 Phe(279) In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe(279), whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe(229).

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.

Hidden States within Disordered Regions of the CcdA Antitoxin Protein.

The bacterial toxin-antitoxin system CcdB-CcdA provides a mechanism for the control of cell death and quiescence. The antitoxin protein CcdA is a homodimer composed of two monomers that each contain a folded N-terminal region and an intrinsically disordered C-terminal arm. Binding of the intrinsically disordered C-terminal arm of CcdA to the toxin CcdB prevents CcdB from inhibiting DNA gyrase and thereby averts cell death. Accurate models of the unfolded state of the partially disordered CcdA antitoxin can therefore provide insight into general mechanisms whereby protein disorder regulates events that are crucial to cell survival. Previous structural studies were able to model only two of three distinct structural states, a closed state and an open state, that are adopted by the C-terminal arm of CcdA. Using a combination of free energy simulations, single-pair Förster resonance energy transfer experiments, and existing NMR data, we developed structural models for all three states of the protein. Contrary to prior studies, we find that CcdA samples a previously unknown state where only one of the disordered C-terminal arms makes extensive contacts with the folded N-terminal domain. Moreover, our data suggest that previously unobserved conformational states play a role in regulating antitoxin concentrations and the activity of CcdA's cognate toxin. These data demonstrate that intrinsic disorder in CcdA provides a mechanism for regulating cell fate.

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.

Escherichia coli antitoxin MazE as transcription factor: insights into MazE-DNA binding.

Toxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environmental stresses. The mazEF module encodes the MazF toxin and its cognate MazE antitoxin. The highly dynamic MazE possesses an N-terminal DNA binding domain through which it can negatively regulate its own promoter. Despite being one of the first TA systems studied, transcriptional regulation of Escherichia coli mazEF remains poorly understood. This paper presents the solution structure of C-terminal truncated E. coli MazE and a MazE-DNA model with a DNA palindrome sequence ∼ 10 bp upstream of the mazEF promoter. The work has led to a transcription regulator-DNA model, which has remained elusive thus far in the E. coli toxin-antitoxin family. Multiple complementary techniques including NMR, SAXS and ITC show that the long intrinsically disordered C-termini in MazE, required for MazF neutralization, does not affect the interactions between the antitoxin and its operator. Rather, the MazE C-terminus plays an important role in the MazF binding, which was found to increase the MazE affinity for the palindromic single site operator.

The Thermodynamic Basis of the Fuzzy Interaction of an Intrinsically Disordered Protein.

Many intrinsically disordered proteins (IDP) that fold upon binding retain conformational heterogeneity in IDP-target complexes. The thermodynamics of such fuzzy interactions is poorly understood. Herein we introduce a thermodynamic framework, based on analysis of ITC and CD spectroscopy data, that provides experimental descriptions of IDP association in terms of folding and binding contributions which can be predicted using sequence folding propensities and molecular modeling. We show how IDP can modulate the entropy and enthalpy by adapting their bound-state structural ensemble to achieve optimal binding. This is explained in terms of a free-energy landscape that provides the relationship between free-energy, sequence folding propensity, and disorder. The observed "fuzzy" behavior is possible because of IDP flexibility and also because backbone and side-chain interactions are, to some extent, energetically decoupled allowing IDP to minimize energetically unfavorable folding.

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.