Cobalt(II) coordination compounds with acetate and 2-aminopyridine ligands: Synthesis, characterization, structures and magnetic properties of two polymorphic forms

The synthesis and characterization of two polymorphic modifications of new cobalt coordination compound with 2-aminopyridine are reported. The modifications were prepared by the reaction of a solution of cobalt acetate tetrahydrate and 2-aminopyridine. The crystal structures of both polymorphic modifications have been determined by single-crystal X-ray diffraction analysis. The structures of both modifications are quite similar. In both of them the Co²⁺ is six coordinated by four O atoms from two bidentate chelate acetate ligands and by two N atoms from two 2-aminopyridine molecules. Acetate and 2-aminopyridine ligands are lying cis about the metal centre. The most important difference is in asymmetry of acetate ligand chelate bonding and in H-bonding network. Compounds exhibit an extensive system of intra and intermolecular hydrogen bonding. Magnetic properties of both modifications were studied between 2 K and 300 K giving the result μ_eff = 4.6 BM for modification I and μ_eff = 4.7 BM for modification II in paramagnetic region.     

Flavonoids and cinnamic acid esters as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase: a synthesis, QSAR and modelling study

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) modulate the biological potency of estrogens and androgens by interconversion of inactive 17-keto-steroids and their active 17beta-hydroxy- counterparts. We have shown previously that flavonoids are potentially useful lead compounds for developing inhibitors of 17beta-HSDs. In this paper, we describe the synthesis and biochemical evaluation of structurally analogous inhibitors, the trans-cinnamic acid esters and related compounds. Additionally, quantitative structure-activity relationship (QSAR) and modelling studies were performed to rationalize the results and to suggest further optimization. The results stress the importance of a hydrogen bond with Asn154 and hydrophobic interactions with the aromatic side chain of Tyr212 for optimal molecular recognition.     

Essential Roles of Hydrophobic Residues in Both MD-2 and Toll-like Receptor 4 in Activation by Endotoxin

Gram-negative bacterial endotoxin (i.e. lipopolysaccharide (LPS)) is one of the most potent stimulants of the innate immune system, recognized by the TLR4·MD-2 complex. Direct binding to MD-2 of LPS and LPS analogues that act as TLR4 agonists or antagonists is well established, but the role of MD-2 and TLR4 in receptor activation is much less clear. We have identified residues within the hairpin of MD-2 between strands five and six that, although not contacting acyl chains of tetraacylated lipid IVa (a TLR4 antagonist), influence activation of TLR4 by hexaacylated lipid A. We show that hydrophobic residues at positions 82, 85, and 87 of MD-2 are essential both for transfer of endotoxin from CD14 to monomeric MD-2 and for TLR4 activation. We also identified a pair of conserved hydrophobic residues (Phe-440 and Phe-463) in leucine-rich repeats 16 and 17 of the TLR4 ectodomain, which are essential for activation of TLR4 by LPS. F440A or F463A mutants of TLR4 were inactive, whereas the F440W mutant retained full activity. Charge reversal of neighboring cationic groups in the TLR4 ectodomain (Lys-388 and Lys-435), in contrast, did not affect cell activation. Our mutagenesis studies are consistent with a molecular model in which Val-82, Met-85, and Leu-87 in MD-2 and distal portions of a secondary acyl chain of hexaacylated lipid A that do not fit into the hydrophobic binding pocket of MD-2 form a hydrophobic surface that interacts with Phe-440 and Phe-463 on a neighboring TLR4·MD-2·LPS complex, driving TLR4 activation.Bacterial lipopolysaccharide (LPS)³ is recognized by the innate immune system of vertebrates via an elaborate mechanism involving the membrane receptor TLR4 (1, 2). The extracellular (or cell surface) proteins LPS-binding protein and CD14 promote extraction and transfer of individual molecules of LPS from the Gram-negative bacterial outer membrane to MD-2, either secreted monomeric soluble (s)MD-2 or MD-2 bound with high affinity to the ectodomain of TLR4 (3–7). In contrast to other Toll-like receptors, TLR4 requires an additional molecule, MD-2, for ligand recognition (8). In contrast to MD-2, there has been no evidence of direct binding of LPS to TLR4 (9, 10). Although LPS, and particularly the lipid A portion of LPS, is generally conserved among Gram-negative bacteria, there are many variables in LPS structure that affect TLR4 activation. Most important is the acylation pattern of the lipid A moiety, which represents the minimal segment of LPS that can trigger activation of TLR4 (11). Comparison of crystal structures of MD-2 with and without bound tetraacylated lipid IVa indicates no significant alteration of the protein fold in the absence or presence of bound ligand (12). It has been proposed that both LPS and MD-2 are key to the different effects of tetra- versus hexaacylated LPS on TLR4 (8, 13, 14). Lipid IVa complexed to murine MD-2 has weak agonist effects on murine TLR4 but acts as a receptor antagonist in the same complex containing human MD-2. Hexaacylated endotoxins complexed to human or murine MD-2 act as potent TLR4 agonists. The crystal structure of the TLR4·MD-2·eritoran complex revealed that MD-2 binds to the N-terminal region of TLR4 (15). It seems likely that for TLR4 activation, there needs to be an additional interaction between two ternary TLR4·MD-2·LPS complexes, which is agonist-dependent (15–17). Because tetraacylated and hexaacylated endotoxins that act, respectively, as TLR4 antagonists and agonists differ only in their acylation pattern, we speculated that hydrophobic protein-lipid A interactions are essential in the agonist properties of hexaacylated lipid A. To pursue this hypothesis, we used molecular modeling to select and test the involvement of solvent-exposed hydrophobic residues of MD-2 and TLR4, which we reasoned could be needed for TLR4 activation. We show by mutagenesis studies that residues on the solvent-exposed hairpin of MD-2 support transfer of endotoxin from CD14 to MD-2 and TLR4 activation only when these sites contain hydrophobic residues. In the ectodomain of TLR4, we have identified two neighboring phenylalanine residues located on the convex face of consecutive leucine rich repeats that are required for LPS-triggered TLR4 activation. From those results and molecular docking, we propose that amino acid side chains of both MD-2 and TLR4 ectodomain form an acyl chain binding site, which envelops part of an acyl chain of lipid A that cannot fit into the binding pocket of MD-2 in a TLR4·MD-2 complex and represents a key to LPS-induced TLR4 activation.     

HWGMWSY, an unanticipated polystyrene binding peptide from random phage display libraries

Phage display is a powerful technique for the discovery of peptide ligands that bind to various targets; however, ambiguous results often appear. Peptide HWGMWSY has been isolated repeatedly in our laboratory and by other research groups dealing with different protein and nonprotein targets, which led to a hypothesis that it may be a target-unrelated peptide interacting with polystyrene plastic surfaces. We compared binding properties and amplification rate of phage clone displaying the peptide HWGMWSY, a previously confirmed plastic binding clone WHWRLPS, and a control phage clone ASVQERK. An enzyme-linked immunosorbent assay and a phage elution assay confirmed that phage clone HWGMWSY binds to polystyrene. Surface plasmon resonance measurements on the other hand excluded the possibility of binding to bovine serum albumin, a common blocking agent in phage display experiments. Amplification rates of the above-noted phage clones were not statistically different. We therefore conclude that phage clone HWGMWSY was isolated in different selection procedures as a result of its affinity to polystyrene.     

Range and local population densities of brown bear Ursus arctos in Slovenia

Solid understanding of species’ range and local population densities is important for successful wildlife management and research. Specific behavioral and ecological characteristics make brown bear Ursus arctos a difficult species to study. We present a map of range and local population densities of brown bears in Slovenia, made with the use of a new approach similar to voting classifications based on a combination of four datasets: Global Positioning System telemetry data, records of bear removals, systematic and opportunistic direct observations and signs of bear presence, and noninvasive genetic samples. Results indicate that the majority of bears in Slovenia live in Dinaric Mountains in the southern part of the country where local bear population densities exceed 40 bears/100 km². This is one of the highest population densities reported so far for this species worldwide. Population densities decrease towards the north (Alpine region) and are very low along the border with Italy and Austria where almost no females are present. This explains slow past and present expansion of this transboundary bear population into the Alps and should be considered in future bear re-colonization management strategies. Results also showed that data from observations and removals overestimate bear population densities at low values, while mortality and genetic data overestimate population densities in areas with more people. Nevertheless, all data types appeared useful for describing the general bear distribution patterns. Similar approach could be applied to studies of other charismatic or game species, for which several types of data are often available.     

Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines

Endosomal TLRs play an important role in innate immune response as well as in autoimmune processes. In the therapy of systemic lupus erythematosus, antimalarial drugs chloroquine, hydroxychloroquine, and quinacrine have been used for a long time. Their suppression of endosomal TLR activation has been attributed to the inhibition of endosomal acidification, which is a prerequisite for the activation of these receptors. We discovered that chloroquine inhibits only activation of endosomal TLRs by nucleic acids, whereas it augments activation of TLR8 by a small synthetic compound, R848. We detected direct binding of antimalarials to nucleic acids by spectroscopic experiments and determined their cellular colocalization. Further analysis revealed that other nucleic acid-binding compounds, such as propidium iodide, also inhibited activation of endosomal TLRs and colocalized with nucleic acids to endosomes. We found that imidazoquinolines, which are TLR7/8 agonists, inhibit TLR9 and TLR3 even in the absence of TLR7 or TLR8, and their mechanism of inhibition is similar to the antimalarials. In contrast to bafilomycin, none of the tested antimalarials and imidazoquinolines inhibited endosomal proteolysis or increased the endosomal pH, confirming that inhibition of pH acidification is not the underlying cause of inhibition. We conclude that the direct binding of inhibitors to nucleic acids mask their TLR-binding epitope and may explain the efficiency of those compounds in the treatment of autoimmune diseases.     

Disulfide mapping reveals the domain swapping as the crucial process of the structural conversion of prion protein

Prion diseases are infectious conformational diseases. Despite the determination of many native prion protein (PrP) structures and in vitro production of infectious prions from recombinant PrP the structural background of PrP conversion remains the largest unsolved problem. The aggregated state of PrP^Sc makes it inaccessible to high resolution techniques, therefore indirect methods have to be used to investigate the conversion process. We engineered disulfide bridges into the structured domain of PrP in order to determine the secondary structure elements that remain conserved upon conversion. Rather surprisingly, introduction of disulfides into each or both of the subdomains B1-H1-B2 and H2-H3 of the C-terminal globular domain retained the robust ability to convert into fibrils with increased content of β-structure, indistinguishable from the wild-type PrP. On the other hand disulfide bridges tethering the two subdomains completely prevented conversion, while their reduction reversed their conversion ability. The same conversion propensity was replicated also in prion infected cell lines. Experiments with combinations of engineered cysteine residues further support that domain swapping, centered on the B2-H2 loop, previously associated to species barrier, leads to PrP swapped dimers as the building block of prion fibrils.Key words: PrP, prion protein, mPrP, murine prion protein, prion protein, structural conversion, disulfide crosslinks, secondary structure, domain swapping, rigid loop, dimerOur understanding of the molecular mechanisms of prion diseases recently significantly advanced with the invention of PMCA technique¹ and the demonstration that the converted recombinant PrP can induce transmissible disease,^2–5 which conclusively fulfills the Koch''s postulates of infectivity. Another important development, relevant to the structural background of conformational diseases was the demonstration of the ability of short peptides to form cross-β-structure in many diverse orientations forming the so called dry steric zippers, which might underlay the existence of different prion strains.⁶ However the main question on the biochemical and structural nature of PrP conversion process remained unanswered.Prion diseases are characterized by conversion of the native PrP into the form PrP^Sc, which forms amyloid aggregates that are resistant to proteolysis. Tertiary structure of the native form of PrP from more than 15 different species has been determined.⁷ Their fold is highly conserved, with an unstructured N-terminal half of the protein and a C-terminal structured domain consisting of three α-helices and two β-strands.⁸ The native form of PrP exhibits high content of α-helical structure, while the converted form is dominated by the β-type secondary structure. The secondary structure content of the PrP^Sc is somewhat controversial. Analyses of infrared or CD spectra suggest that the secondary structure of converted PrP contains between 17–30% of α-helix and 43–50% of β-structure.^9,10 This is clearly different from the all-β structure. It is in fact compatible with the conservation of the large part of the secondary structure elements of the C-terminal globular domain and induced formation of the β-structure from the proximal N-terminal segment, disordered in the native state.The defined tertiary structure of proteins is determined by the multitude of cooperative interactions that provide the sufficient free energy gap between the native and nonnative conformations. The existence of alternative, significantly different global folds of any protein has not been demonstrated yet at the level of a defined tertiary structure. It would be in fact extremely difficult to stabilize the alternative stable fold, where many of the corporative interactions would have to be optimized simultaneously. This proposition is supported by the observation that most of the proteins involved in conformational diseases contain a segment with an intrinsically unfolded structure in the native state.¹¹ Therefore it is much more likely for those unfolded segments to adopt an ordered conformation rather than to completely refold the native globular structure.Aggregation state of the PrP^Sc hinders determination of high resolution structure. We can however use different biochemical approaches to inquire about the nature of the conversion process and structure of the converted form. Methods, such as antibody mapping,^12,13 hydrogen exchange,^14–16 binding of fluorescent ligands¹⁷ and many others have been used, revealing that both C-terminal and proximal N-terminal segment of the PrP become less accessible to the solvent upon conversion.In order to unravel the molecular mechanism of PrP conversion, we decided to investigate which of the secondary structure elements or their suprasecondary structure combinations are retained in the converted form. We introduced disulfide tethers into different positions within the globular C-terminal segment of mPrP, connecting different secondary structure elements.¹⁸ Several pairs of residues that adhere to the geometric requirements for a disulfide formation were selected.¹⁹ Covalent tethers impose a very strong structural constraint as the relative position of the tethered pair needs to remain the same in the converted structure. This approach therefore allowed us to probe the relative position of all secondary structure elements in the converted PrP. We successfully prepared seven disulfide-tethered variants of mPrP. The only variants that we could not prepare were those where the introduced cysteines were in the neighborhood of the existing disulfide and probably led to the heterogeneous disulfide shuffling yielding misfolded products. We demonstrated that in all variants additional disulfides are formed and the secondary structure of the native form of PrP variants is indistinguishable from the wild-type PrP.The key experiment was in vitro conversion of PrP disulfide mutants. Surprisingly, the majority of the disulfide-tethered variants was able to convert into PrP fibrils. Mutant fibrils had the same morphology, determined by AFM and TEM, pattern of antibody mapping and high content of β-structure as the fibrils prepared from wild-type PrP. The common structural property of the three variants that did not convert and retained the native, α-helical conformation, regardless of the conversion protocol, is that they all tethered the two subdomains B1-H1-B2 and H2-H3 to each other (Fig. 1). The proof that this is indeed an intrinsic structural property rather than a result of serendipitous point mutations is that both variants with single cysteine residues of the disulfide pair retained the ability to convert. Moreover reduction of disulfides rendered the originally non-converting disulfide variants convertible into the β-structured fibrils. Even simultaneous introduction of two new disulfides, one into each of the two subdomains, retained the ability of PrP variants to convert. Introduction of disulfides predominantly improved the stability of the protein, increasing the Tm by 3–12 degrees. However, the conversion ability had no correlation with the thermal stability of the protein as some of the most stable variants, containing two disulfides that increased Tm by more than 16 degrees, readily converted.Open in a separate windowFigure 1Mapping of PrP conversion by disulfide tethers. Disulfides engineered within the globular domain of PrP have different effects on its ability to convert into fibrils. Disulfide tethers are schematically represented as straight connectors on mouse PrP structure (1XYX).²² All disulfides (left top), which tether on one side subdomain B1-H1-B2 (gray) and on the other subdomain H2-H3 (black) prevent conversion, while PrP variants with single or even double disulfide tethers within each or both of the two subdomains retain the ability to convert into fibrils (left bottom). Results suggest that the secondary structure of each of the two subdomains is conserved during conversion, which can be accomplished by separation of subdomains (middle) followed by domain swapping. Domain swapped PrP dimer thus represents the building block of fibrils and a template for the annealing of the disordered N-terminal part into β-structure. Monomers within a swapped dimer are shown in gray and black (right).Those results provide an exceptionally strong set of constraints to characterize the conversion process and structure of the converted form. Our results are not compatible with most of the current structural models of PrP conversion, which suggest unfolding or significant rearrangements of secondary structure elements of the globular domain.^14,16,20,21 Separation of subdomains of PrP implies that this process requires high activation energy or highly unfolding conditions. The loop linking the two subdomains connects B2 to H2 and has also been called “the rigid loop,” named by the increased ordering in the elk PrP in contrast to mouse or human PrP.²² This loop has been implicated in the species barrier^23,24 and protective polymorphisms.²⁵ Mice carrying mutations S170N N174T, where residues from mouse PrP are replaced with the corresponding residues from elk, develop spontaneous transmissible prion disease.²⁶It might be in principle possible that disulfide variants convert off-pathway from the physiologically relevant PrP^Sc form. However we were able to demonstrate the same properties in cell cultures; only in vitro convertible PrP variant was able to replicate prions, while the unconvertible variant did not.The only structural transition that is compatible with our results is domain swapping of the C-terminal globular domain. Domain swapping represents the mechanism of oligomerization where the monomer and oligomer share the majority of the secondary structure elements. Most of the residues in the swapped-dimer oligomer are in exactly the same type of chemical environment as in the monomer with the exception of residues that represent the hinge of subdomain separation and connection between the monomeric units. Domain swapping requires high activation energy as the monomer has to unfold during conversion. The resulting oligomers are typically extremely stable and often a single protein can form different domain-swapped oligomers.²⁷In order to confirm domain-swapped model of prion protein conversion we performed additional experiments where we analyzed the conversion products of a mixture of the two single cysteine mutants. Those single cysteine variants were designed in a way that if swapping of the sub-domains B1-H1-B2 and H2-H3 occurs during conversion, cysteines from different single cysteine variants come into contact and can form a disulfide bridge. Indeed proteinase K-resistant covalent dimers were only observed upon conversion of a mixture of both variants.In conclusion, we present the model of PrP conversion, where the conversion process requires unfolding of the core of the structured C-terminal domain of PrP with separation of the two subdomains, which recombine into a swapped dimer (Fig. 1). It has been demonstrated previously by several different approaches that PrP dimerization is important and a rate limiting step in conversion.^28–30 This swapped dimer represents the building block of fibrils and the template for structuring of the unfolded N-terminal segment, which can anneal to the dimer in the form of the β-strands, such as demonstrated in peptide dry steric zippers. We propose that the variability between different strains of prions may originate from differently annealed β-strands of the N-terminal segments and can additionally be affected by posttranslational modifications and the presence of additional molecules, such as nucleic acids or lipids.     

Functional self-assembling polypeptide bionanomaterials

Bionanotechnology seeks to modify and design new biopolymers and their applications and uses biological systems as cell factories for the production of nanomaterials. Molecular self-assembly as the main organizing principle of biological systems is also the driving force for the assembly of artificial bionanomaterials. Protein domains and peptides are particularly attractive as building blocks because of their ability to form complex three-dimensional assemblies from a combination of at least two oligomerization domains that have the oligomerization state of at least two and three respectively. In the present paper, we review the application of polypeptide-based material for the formation of material with nanometre-scale pores that can be used for the separation. Use of antiparallel coiled-coil dimerization domains introduces the possibility of modulation of pore size and chemical properties. Assembly or disassembly of bionanomaterials can be regulated by an external signal as demonstrated by the coumermycin-induced dimerization of the gyrase B domain which triggers the formation of polypeptide assembly.     

The Lipopolysaccharide Core of Brucella abortus Acts as a Shield Against Innate Immunity Recognition

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.