Cytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings ...localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ.
Significance Division of bacteria is executed by a contractile ring whose cytoskeletal framework is FtsZ (filamentation temperature-sensitive Z), a protein evolutionarily related to eukaryotic tubulin. The FtsZ ring is made of filaments of head-to-tail FtsZ subunits but its architecture and the rules governing its assembly are still poorly known. Here we show that MciZ, an inhibitor of FtsZ ring formation, functions by capping the minus end of FtsZ filaments. Capping by MciZ makes FtsZ filaments shorter than normal, likely by blocking filament annealing; this represents fundamental information to understand how FtsZ filaments grow and shrink, and attain their normal size. The powerful inhibition of Z-ring assembly by MciZ also suggests that an FtsZ ring cannot form from filaments smaller than a certain size.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The antibacterial activity of a calixarene derivative, p-tert-butylcalix6arene (Calix6), was assessed and was shown not to inhibit the growth of E. coli, S. aureus and B. subtilis bacteria. With the ...aim of gaining more insights into the absence of antibacterial activity of Calix6, the interaction of this derivative with DPPG, a bacterial cell membrane lipid, was studied. Langmuir monolayers were used as the model membrane. Pure DPPG and pure Calix6 monolayers, as well as binary DPPG:Calix6 mixtures were studied using surface pressure measurements, compressional modulus, Brewster angle and fluorescence microscopies, ellipsometry, polarization-modulation infrared reflection absorption spectroscopy and molecular dynamics simulations. Thermodynamic properties of the mixed monolayers were additionally calculated using thermodynamic parameters. The analysis of isotherms showed that Calix6 significantly affects the DPPG monolayers, modifying the isotherm profile and increasing the molecular area, in agreement with the molecular dynamics simulations. The presence of Calix6 in the mixed monolayers decreased the interfacial elasticity, indicating that calixarene disrupts the strong intermolecular interactions of DPPG hindering its organization into a compact arrangement. At low molar ratios of Calix6, the DPPG:Calix6 interactions are preferentially attractive, due to the interactions between the hydrophobic tails of DPPG and the tert-butyl groups of Calix6. Increasing the proportion of calixarene generates repulsive interactions. Calix6 significantly affects the hydrophobic tail organization, which was confirmed by PM-IRRAS measurements. Calix6 appears to be expelled from the mixed films at a biologically relevant surface pressure, π = 30 mN m-1, indicating a low interaction with the cell membrane model related to the absence of antibacterial activity.
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Ruthenium-based anticancer agents have created a center of attention in the field of inorganic medicinal chemistry. The first fully characterized cationic ruthenium(II)-arene ...complexes Ru(η6-p-cymene) (PAr3)LNCl+ with highly lipophilic PAr3 ligands where Ar = 3,5-((CH3)3C)2C6H3– (L1), 3,5-(CH3)2C6H3– (L2), 4-CH3O-3,5-(CH3)2C6H2– (L3) and 4-CH3O-C6H4– (L4) with N = 3-methylpyridine (1–4, respectively), or L4 and 4-methylpyridine (5), or L4 and CH3CN (6) were obtained (yields 67–91%) as solids stable to light and air. Electrical conductance indicates that all the complexes are 1:1 electrolytes in solution. Their composition and purity have been unambiguously established by single-crystal X-ray diffraction, NMR spectroscopy and elemental analysis. The coordination geometries are uniform for all six complexes and each structure consist of a unipositive complex cation bearing the phosphine ligands L1-L4 and LN = 3-methylpyridine, 4-methylpyridine or CH3CN attached to the organometallic fragment. The equivalent unit cell volumes per formula unit decrease with 1 > 3 > 2 > 4 > 5 > 6, accurately reflecting the decreasing sizes of the phosphines L1-L4, and a greater occupied volume for 3-methyl- vs. 4-methylpyridine, and the smallest volume contribution from CH3CN. Electrochemical studies showed mixed electrochemical mechanisms (EC/ECE) from partial substitution of p-cymene by CH3CN ligands from the solvent. A large electrochemical stability window (>2.2 V) for Ru(II) was observed extending beyond the physiological E° range. The complexes were cytotoxic against human cancer cell lines in vitro, and some complexes altered cell morphology.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•Chalcones evaluated presented promising schistosomicidal effects.•Chalcones evaluated have a conformational preference for s-cis geometry.•The ortho-substituents chalcones presented possible ...relation between s-trans geometries with schistosomicidal effects.•NMR spectroscopy was used for characterization.
Chalcones are compounds associated to several biological activities. The modification of chemical structures is one of the most widely used tool in the pharmaceutical industry to obtain new drugs. In this study, we synthetized chalcones with nitro and bromo substituents in different positions in the aromatic rings. NMR spectroscopy was used for characterization. Simultaneously, a conformational study of the compounds was carried out employing theoretical calculations. Furthermore, the schistosomicidal in vitro effect of bromo and nitro chalcone substituents on adult Schistosoma mansoni was investigated. The results showed that most chalcones have a conformational preference for s-cis geometry, which is favored by the conjugative effect of electronic delocalization. However, chalcones with ortho substituents dissipate greater stabilization of s-trans conformations, possibly for favoring surfaces with weak interactions such as Van der Waals. In addition, a significant effect of chalcones on female and male S. mansoni was observed. All samples evaluated reduced the motor activity of worms, caused mortality in male and female worms, and resulted in morphological alterations in a concentration- dependent manner (25–200 μg/mL) in a period of 6–24 h of incubation. Additionally, chalcones influenced the separation of couple worms and suppressed egg production at sublethal concentrations. Our results demonstrated that chalcones present potential to be used as schistosomicidal drugs, contributing to the clarification of the relation between the molecule conformation and the associated biological effects.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The synthesis and spectroscopic characterization of four ruthenium(II) arene complexes with monodentate pyridine derivatives ((η6–p-cymene)RuCl2L: L = 2-aminopyridine, 2-methylaminopyridine, ...2-benzylaminopyridine, and pyridine) are reported. Full characterization was undertaken using 1H and 13C NMR spectroscopy, vibrational and electronic spectroscopies and crystallography (2-methylaminopyridine derivative). UB3LYP//(6-31 + G(d),SPK-DZCD) density functional theory calculations determined the molecular and electronic structures. Cyclic voltammetry determined a large electrochemical stability window (>2.2 V) extending well beyond the physiological E°. Interactions with CT-DNA and BSA, and activity against four cell lines (HeLa, B16F10, HEp-2 and Vero) were evaluated. The 2-methylaminopyridine shows weak cytotoxicity (IC50 = 346 μmol L−1) towards HeLa cells. All the complexes interact with DNA at relatively high concentrations as determined by UV–vis spectroscopic titration. Results of circular dichroism spectroscopy, ethidium bromide competition, fluorescence spectroscopy and DNA viscosity measurements identify electrostatic interactions between partly hydrolyzed cationic complexes and the phosphate backbone of DNA as the most likely interaction mode. Slower rates of hydrolysis may be the origin of lower cytotoxicity for κ1 these complexes.
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•Four (η6–p-cymene)RuCl2L complexes with monodentate pyridine ligands were synthetized.•Crystal structure of the complex with L= 2-methylaminopyridine displays a ligand N–H to metal-bound chloride hydrogen bonds.•The lack of cytotoxic activity against HeLa cell line could be attributed to electrostatic interactions.•A large electrochemical stability window these complexes extending beyond the physiological E° range.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
FtsZ is a self-assembling GTPase that forms, below the inner membrane, the mid-cell Z-ring guiding bacterial division. FtsZ monomers polymerize head to tail forming tubulin-like dynamic ...protofilaments, whose organization in the Z-ring is an unresolved problem. Rather than forming a well-defined structure, FtsZ protofilaments laterally associate in vitro into polymorphic condensates typically imaged on surfaces. We describe here nanoscale self-organizing properties of FtsZ assemblies in solution that underlie Z-ring assembly, employing time-resolved x-ray scattering and cryo-electron microscopy. We find that FtsZ forms bundles made of loosely bound filaments of variable length and curvature. Individual FtsZ protofilaments further bend upon nucleotide hydrolysis, highlighted by the observation of some large circular structures with 2.5–5° curvature angles between subunits, followed by disassembly end-products consisting of highly curved oligomers and 16-subunit −220 Å diameter mini-rings, here observed by cryo-electron microscopy. Neighbor FtsZ filaments in bundles are laterally spaced 70 Å, leaving a gap in between. In contrast, close contact between filament core structures (∼50 Å spacing) is observed in straight polymers of FtsZ constructs lacking the C-terminal tail, which is known to provide a flexible tether essential for FtsZ functions in cell division. Changing the length of the intrinsically disordered C-tail linker modifies the interfilament spacing. We propose that the linker prevents dynamic FtsZ protofilaments in bundles from sticking to one another, holding them apart at a distance similar to the lateral spacing observed by electron cryotomography in several bacteria and liposomes. According to this model, weak interactions between curved polar FtsZ protofilaments through their the C-tails may facilitate the coherent treadmilling dynamics of membrane-associated FtsZ bundles in reconstituted systems, as well as the recently discovered movement of FtsZ clusters around bacterial Z-rings that is powered by GTP hydrolysis and guides correct septal cell wall synthesis and cell division.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cell division in bacteria is regulated by proteins that interact with FtsZ and modulate its ability to polymerize into the Z ring structure. The best studied of these regulators is MinC, an inhibitor ...of FtsZ polymerization that plays a crucial role in the spatial control of Z ring formation. Recent work established that E. coli MinC interacts with two regions of FtsZ, the bottom face of the H10 helix and the extreme C-terminal peptide (CTP). Here we determined the binding site for MinC on Bacillus subtilis FtsZ. Selection of a library of FtsZ mutants for survival in the presence of Min overexpression resulted in the isolation of 13 Min-resistant mutants. Most of the substitutions that gave rise to Min resistance clustered around the H9 and H10 helices in the C-terminal domain of FtsZ. In addition, a mutation in the CTP of B. subtilis FtsZ also produced MinC resistance. Biochemical characterization of some of the mutant proteins showed that they exhibited normal polymerization properties but reduced interaction with MinC, as expected for binding site mutations. Thus, our study shows that the overall architecture of the MinC-FtsZ interaction is conserved in E. coli and B. subtilis. Nevertheless, there was a clear difference in the mutations that conferred Min resistance, with those in B. subtilis FtsZ pointing to the side of the molecule rather than to its polymerization interface. This observation suggests that the mechanism of Z ring inhibition by MinC differs in both species.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•A rationally selected series of PAr3 more basic than PPh3.•Phosphines enhance the lipophilicity of neutral η6-arene Ru complexes.•Langmuir films demonstrate how geometries are dominated by bulky ...phosphines.•Redox-stability window exceeding the reduction and oxidation of aqueous media.•Cytotoxicity against healthy and cancerous cell lines shows partial selectivity.
Neutral Ru(II)-arene complexes Ru(η6‐p-cymene)(PAr3)Cl2, PAr3 where Ar = 3,5-((CH3)3C)2C6H3–, 3,5-(CH3)2C6H3–, 4-CH3O-3,5-(CH3)2C6H2– and 4-CH3O-C6H4– were synthetized and fully characterized including single-crystal X-ray diffraction. The ligands have empirical logPow = 10.29, 7.38, 7.08, 5.43 (vs. PPh3 = 5.14). They have sufficiently low solubility to form Langmuir monolayers on water in accordance with overall molecular sizes. Density functional theory (DFT) calculations were performed to determine the molecular and electronic structures and to interpret the results of voltammetry and UV–Vis spectroscopy. Cytotoxicity assays were evaluated against MRC-5 (non-tumoral lung), A549 (lung cancer) and MDA-MB-231 (breast cancer) cell lines. The complexes with Ar = 3,5-((CH3)3C)2C6H3–, 4-CH3O-3,5-(CH3)2C6H2– and 4-CH3O-C6H4– show high cytotoxicity, whilst the first complex is inactive towards the (healthy) MRC-5 cell line, indicating a degree of selectivity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The antibacterial activity of a calixarene derivative,
p-tert
-butylcalix6arene (
Calix6
), was assessed and was shown not to inhibit the growth of
E. coli
,
S. aureus
and
B. subtilis
bacteria. With ...the aim of gaining more insights into the absence of antibacterial activity of
Calix6
, the interaction of this derivative with DPPG, a bacterial cell membrane lipid, was studied. Langmuir monolayers were used as the model membrane. Pure DPPG and pure
Calix6
monolayers, as well as binary DPPG:
Calix6
mixtures were studied using surface pressure measurements, compressional modulus, Brewster angle and fluorescence microscopies, ellipsometry, polarization-modulation infrared reflection absorption spectroscopy and molecular dynamics simulations. Thermodynamic properties of the mixed monolayers were additionally calculated using thermodynamic parameters. The analysis of isotherms showed that
Calix6
significantly affects the DPPG monolayers, modifying the isotherm profile and increasing the molecular area, in agreement with the molecular dynamics simulations. The presence of
Calix6
in the mixed monolayers decreased the interfacial elasticity, indicating that calixarene disrupts the strong intermolecular interactions of DPPG hindering its organization into a compact arrangement. At low molar ratios of
Calix6
, the DPPG:
Calix6
interactions are preferentially attractive, due to the interactions between the hydrophobic tails of DPPG and the
tert
-butyl groups of
Calix6
. Increasing the proportion of calixarene generates repulsive interactions.
Calix6
significantly affects the hydrophobic tail organization, which was confirmed by PM-IRRAS measurements.
Calix6
appears to be expelled from the mixed films at a biologically relevant surface pressure,
π
= 30 mN m
−1
, indicating a low interaction with the cell membrane model related to the absence of antibacterial activity.
Expulsion of
p-tert
-butylcalix6arene molecules from a monolayer in a biologically relevant pressure regime,
π
= 30 mN m
−1
, correlates with their lack of antibacterial activity.
The
Azospirillum brasilense genomic region containing the
glnBA genes was previously cloned in a broad-host range plasmid named pAB441. Previous results from our laboratory have shown that
A. ...brasilense spontaneous mutants resistant to ethylenediamine carrying this plasmid had no nitrogenase activity. The 19
kb insert of this plasmid was fully sequenced and no other gene potentially involved with nitrogen fixation was identified, nor any gene potentially involved in the regulation of
nif genes expression. Apart from
glnBA, this region contains several genes involved with different functions: assisting protein folding and degradation, DNA organization, electron transport and energy conversion and conserved hypothetical proteins of unknown function. Since previous data on the literature showed that mutation of
clpX gene of
A. brasilense resulted in higher nitrogenase activity, we hypothesize that higher expression of the
clpX gene from the plasmid pAB441 may be involved with Nif
− phenotype of the
A. brasilense mutant strains.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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