Crystal structure of the intrinsically flexible addiction antidote MazE

A specific camel VHH (variable domain of dromedary heavy chain antibody) fragment was used to crystallize the intrinsically flexible addiction antidote MazE. Only 45% of the polypeptide chain is found ordered in the crystal. The MazE monomer consisting of two beta-hairpins connected by a short alpha-helix has no hydrophobic core on its own and represents only one half of a typical protein domain. A complete domain structure is formed by the association of two chains, creating a hydrophobic core between two four-stranded beta-sheets. This hydrophobic core consists exclusively of short aliphatic residues. The folded part of MazE contains a novel DNA binding motif. A model for DNA binding that is consistent with the available biochemical data is presented.     

Recognition of the intrinsically flexible addiction antidote MazE by a dromedary single domain antibody fragment. Structure,thermodynamics of binding,stability, and influence on interactions with DNA

The Escherichia coli mazEF operon defines a chromosomal addiction module that programs cell death under various stress conditions. It encodes the toxic and long-lived MazF and the labile antidote MazE. The denaturation of MazE is a two-state reversible dimer-monomer transition. At lower concentrations the denatured state is significantly populated. This leads to a new aspect of the regulation of MazE concentration, which may decide about the life and death of the cell. Interactions of MazE with a dromedary antibody domain, cAbMaz1 (previously used as a crystallization aid), as well as with promoter DNA were studied using microcalorimetric and spectroscopic techniques. Unique features of cAbMaz1 enable a specific enthalpy-driven recognition of MazE and, thus, a significant stabilization of its dimeric native conformation. The MazE dimer and the MazE dimer-cAbMaz1 complex show very similar binding characteristics with promoter DNA, i.e. three binding sites with apparent affinities in micromolar range and highly exothermic binding accompanied by large negative entropy contributions. A working model for the MazE-DNA assembly is proposed on the basis of the structural and binding data. Both binding and stability studies lead to a picture of MazE solution structure that is significantly more unfolded than the structure observed in a crystal of the MazE-cAbMaz1 complex.     

Specific potassium binding stabilizes pI258 arsenate reductase from Staphylococcus aureus

Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 catalyzes the reduction of arsenate to arsenite and plays a role in bacterial heavy metal resistance. The high resolution x-ray structure of ArsC reveals the atomic details of the K+ binding site situated next to the catalytic P-loop structural motif of this redox enzyme. A full thermodynamic study of the binding characteristics of a series of monovalent cations (Li+, Na+, K+, Rb+, and Cs+) and their influence on the thermal stability of ArsC was performed with isothermal titration calorimetry, circular dichroism spectroscopy, and differential scanning calorimetry. Potassium has the largest affinity with a Ka of 3.8 x 10(3) m(-1), and the effectiveness of stabilization of ArsC by monovalent cations follows the binding affinity order: K+ > Rb+ > Cs+ > Na+ > Li+. A mutagenesis study on the K+ binding side chains showed that Asn-13 and Asp-65 are essential for potassium binding, but the impact on the stability of ArsC was the most extreme when mutating Ser-36. Additionally, the thermal stabilization by K+ is significantly reduced in the case of the ArsC E21A mutant, showing the importance of a Glu-21-coordinated water molecule in its contact with K+. Although potassium is not essential for catalysis, in its presence the kcat/KM increases with a factor of 5. Altogether, the interaction of K+ with specific residues in ArsC is an enthalpydriven process that stabilizes ArsC and increases the specific activity of this redox enzyme.     

Adaptation of adhesion test using Caco-2 cells for anaerobic bacterium<Emphasis Type="Italic">Pseudobutyrivibrio xylanivorans</Emphasis>, a probiotic candidate

Pseudobutyrivibrio xylanivorans strain Mz5(T), an anaerobic bacterium (originating from the rumen of a Holstein-Friesian cow), has some attributes that make it a possible probiotic strain (very active hydrolases, bacteriocin and conjugated linoleic acid production). For the estimation of its adhesion ability, the adhesion test on Caco-2 cells was introduced and adapted. The adhesion was performed in an anaerobic glove box in standard 24-well plates at neutral pH for 30 min. The best method for separation of the adhered bacteria from Caco-2 cells appeared to be homogenization with an automatic pipette. The number of adhered bacteria was too small to be determined microscopically, so a new approach, i.e. detection of the apparent lag phase in liquid growth medium was tested. Under the selected assay conditions 1.04 bacterial cells from the late exponential phase adhered to one Caco-2 cell, which confirms the adhesion capability of P. xylanivorans Mz5(T). The adapted adhesion test using Caco-2 cells is suitable for estimation of adhesion capability of anaerobic bacteria.     

Dominant Driving Forces in Human Telomere Quadruplex Binding-Induced Structural Alterations

Recently various pathways of human telomere (ht) DNA folding into G-quadruplexes and of ligand binding to these structures have been proposed. However, the key issue as to the nature of forces driving the folding and recognition processes remains unanswered. In this study, structural changes of 22-mer ht-DNA fragment (Tel22), induced by binding of ions (K⁺, Na⁺) and specific bisquinolinium ligands, were monitored by calorimetric and spectroscopic methods and by gel electrophoresis. Using the global model analysis of a wide variety of experimental data, we were able to characterize the thermodynamic forces that govern the formation of stable Tel22 G-quadruplexes, folding intermediates, and ligand-quadruplex complexes, and then predict Tel22 behavior in aqueous solutions as a function of temperature, salt concentration, and ligand concentration. On the basis of the above, we believe that our work sets the framework for better understanding the heterogeneity of ht-DNA folding and binding pathways, and its structural polymorphism.     

Structural Basis of Epitope Recognition by Heavy-Chain Camelid Antibodies

Truncated versions of heavy-chain antibodies (HCAbs) from camelids, also termed nanobodies, comprise only one-tenth the mass of conventional antibodies, yet retain similar, high binding affinities for the antigens. Here we analyze a large data set of nanobody–antigen crystal structures and investigate how nanobody–antigen recognition compares to the one by conventional antibodies. We find that nanobody paratopes are enriched in aromatic residues just like conventional antibodies, but additionally, they also bear a more hydrophobic character. Most striking differences were observed in the characteristics of the antigen's epitope. Unlike conventional antibodies, nanobodies bind to more rigid, concave, conserved and structured epitopes enriched with aromatic residues. Nanobodies establish fewer interactions with the antigens compared to conventional antibodies, and we speculate that high binding affinities are achieved due to less unfavorable conformational and more favorable solvation entropy contributions. We observed that interactions with antigen are mediated not only by three CDR loops but also by numerous residues from the nanobody framework. These residues are not distributed uniformly; rather, they are concentrated into four structurally distinct regions and mediate mostly charged interactions. Our findings suggest that in some respects nanobody–antigen interactions are more similar to the general protein–protein interactions rather than antibody–antigen interactions.     

Repulsive guidance molecule A (RGMa): A molecule for all seasons

RGMa (repulsive guidance molecule a) was the first identified molecule that possessed the necessary functional activity to repulse and steer growth cones to their target in the brain. By binding to its neogenin receptor, RGMa caused the collapse of growth cones and encouraged axons to grow along specific trajectories in vitro. Although originally characterized in 1990, RGMa was not conclusively shown to mediate axon guidance in vivo for another 12 years. Loss-of-function analysis in mice revealed that RGMa may play a more important role in neural tube morphogenesis. RGMa has now emerged as a molecule with pleiotropic roles involving cell adhesion, cell migration, cell polarity and cell differentiation which together strongly influence early morphogenetic events as well as immune responses. RGMa can be regarded as a molecule for all seasons.     

Kinetically governed polymorphism of d(G₄T₄G₃) quadruplexes in K+ solutions

It has been generally recognized that understanding the molecular basis of some important cellular processes is hampered by the lack of knowledge of forces that drive spontaneous formation/disruption of G-quadruplex structures in guanine-rich DNA sequences. According to numerous biophysical and structural studies G-quadruplexes may occur in the presence of K(+) and Na(+) ions as polymorphic structures formed in kinetically governed processes. The reported kinetic models suggested to describe this polymorphism should be considered inappropriate since, as a rule, they include bimolecular single-step associations characterized by negative activation energies. In contrast, our approach in studying polymorphic behavior of G-quadruplexes is based on model mechanisms that involve only elementary folding/unfolding transitions and structural conversion steps that are characterized by positive activation energies. Here, we are investigating a complex polymorphism of d(G(4)T(4)G(3)) quadruplexes in K(+) solutions. On the basis of DSC, circular dichroism and UV spectroscopy and polyacrylamide gel electrophoresis experiments we propose a kinetic model that successfully describes the observed thermally induced conformational transitions of d(G(4)T(4)G(3)) quadruplexes in terms of single-step reactions that involve besides single strands also one tetramolecular and three bimolecular quadruplex structures.     

The versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.