Structures of amphotericin B-cholesterol complex.

The structures of amphotericin B-cholesterol complex that forms a channel in a lipid membrane were analysed by molecular mechanics calculations. The symmetric complex consisting of eight rigid antibiotic and cholesterol molecules was considered. The presence of a continuous set of low-energy states of the complex with different values of the channel diameter was shown. These states are characterized by significant tilt of the amphotericin planes to the radial axis of the channel and by strong interaction between the charged ammonium and carboxyl groups of the antibiotic. Changes of the channel diameter may result in changes in pore permeability.     

Nuclear calcium and the regulation of the nuclear pore complex

In eukaryotic cells the nucleus and its contents are separated from the cytoplasm by the nuclear envelope. Macromolecules, as well as smaller molecules and ions, can cross the nuclear envelope through the nuclear pore complex. Molecules greater than approx. 60 kDa and containing a nuclear localization signal are actively transported across the nuclear membranes, but there has been little evidence for regulatory mechanisms for smaller molecules and ions. Recently, diffusion across the nuclear envelope has been observed to be regulated by nuclear cisternal Ca²⁺ concentrations. Following depletion of Ca²⁺ from the nuclear store by inositol 1,4,5-trisphosphate or Ca²⁺ chelators, a fluorescent 10 kDa marker molecule was no longer able to enter the nucleus. Distinct conformational states of the nuclear pore complexes depended on the Ca²⁺ filling state of the nuclear envelope, supporting the assumption that a switch in the conformation of the nuclear pore complex may control the transport of intermediate-sized molecules across the nuclear envelope. Thus nuclear Ca²⁺ stores may regulate the conformational state of the nuclear pore complex, and thereby passive diffusion of molecules between the cytosol and the nucleoplasm. The physiological significance of this finding is currently unknown.     

Assembly and stability of nisin-lipid II pores

The peptide antibiotic nisin was the first reported example of an antibiotic that kills bacteria via targeted pore formation. The specific target of nisin is Lipid II, an essential intermediate in the bacterial cell-wall synthesis. High-affinity binding of the antibiotic to Lipid II is followed by rapid permeabilization of the membrane. Here, we investigated the assembly and stability of nisin-Lipid II pore complexes by means of pyrene fluorescence and circular dichroism. We demonstrated that nisin uses all available Lipid II molecules in the membrane to form pore complexes. The pore complexes have a uniform structure and consist of 8 nisin and 4 Lipid II molecules. Moreover, the pores displayed a remarkable stability, because they were able to resist the solubilization of the membrane environment by mild detergents. Similar experiments with [N20P/M21P]nisin showed that the hinge region is essential for the assembly into stable pore complexes. The new insights were used to propose a refined model for nisin pore formation.     

Molecular dynamics simulations of hydrophilic pores in lipid bilayers

Hydrophilic pores are formed in peptide free lipid bilayers under mechanical stress. It has been proposed that the transport of ionic species across such membranes is largely determined by the existence of such meta-stable hydrophilic pores. To study the properties of these structures and understand the mechanism by which pore expansion leads to membrane rupture, a series of molecular dynamics simulations of a dipalmitoylphosphatidylcholine (DPPC) bilayer have been conducted. The system was simulated in two different states; first, as a bilayer containing a meta-stable pore and second, as an equilibrated bilayer without a pore. Surface tension in both cases was applied to study the formation and stability of hydrophilic pores inside the bilayers. It is observed that below a critical threshold tension of approximately 38 mN/m the pores are stabilized. The minimum radius at which a pore can be stabilized is 0.7 nm. Based on the critical threshold tension the line tension of the bilayer was estimated to be approximately 3 x 10(-11) N, in good agreement with experimental measurements. The flux of water molecules through these stabilized pores was analyzed, and the structure and size of the pores characterized. When the lateral pressure exceeds the threshold tension, the pores become unstable and start to expand causing the rupture of the membrane. In the simulations the mechanical threshold tension necessary to cause rupture of the membrane on a nanosecond timescale is much higher in the case of the equilibrated bilayers, as compared with membranes containing preexisting pores.     

Ion coordination in the amphotericin B channel.

The antifungal polyene antibiotic amphotericin B forms channels in lipid membranes that are permeable to ions, water, and nonelectrolytes. Anion, cation, and ion pair coordination in the water-filled pore of the "barrel" unit of the channels was studied by molecular dynamics simulations. Unlike the case of the gramicidin A channel, the water molecules do not create a single-file configuration in the pore, and some cross sections of the channel contain three or four water molecules. Both the anion and cation are strongly bound to ligand groups and water molecules located in the channel. The coordination number of the ions is about six. The chloride has two binding sites in the pore. The binding with water is dominant; more than four water molecules are localized in the anion coordination sphere. Three motifs of the ion coordination were monitored. The dominant motif occurs when the anion is bound to one ligand group. The ion is bound to two or three ligand groups in the less favorable configurations. The strong affinity of cations to the channel is determined by the negatively charged ligand oxygens, whose electrostatic field dominates over the field of the hydrogens. The ligand contribution to the coordination number of the sodium ion is noticeably higher than in the case of the anion. As in the case of the anion, there are three motifs of the cation coordination. The favorable one occurs when the cation is bound to two ligand oxygens. In the less favorable cases, the cation is bound to three or four oxygens. In the contact ion pair, the cation and anion are bound to two ligand oxygens and one ligand hydrogen, respectively. There exist intermediate solvent-shared states of the ion pair. The average distances between ions in these states are twice as large as that of the contact ion pair. The stability of the solvent-shared state is defined by the water molecule oriented along the electrostatic field of both ions.     

Studies by means of 1H NMR spectroscopy of complex formation of aromatic biologically active compounds with antibiotic topotecan

The analysis of heteroassociation of antibiotic topotecan (TPT) with aromatic biologically active compounds (BAC): caffeine, mutagens ethidium bromide and proflavine, antibiotic daunomycin, vitamins flavin-mononucleotide and nicotinamide, has been carried out in the work using 1H NMR spectroscopy data. The equilibrium constants of heteroassociation and induced chemical shifts of the protons have been obtained in the complexes with BAC. It is found that the complex formation TPT-BAC has the nature of stacking of the chromophores, additionally stabilized in the case of proflavine by intermolecular hydrogen bond. Calculation of the basic components of the Gibbs free energy of the complexation reactions is carried out, and the factors which stabilize and destabilize the heterocomplexes of molecules are revealed.     

The selectivity filter of voltage-dependent channels formed by phosphoporin (PhoE protein) from E. coli.

Phosphoporin, an Escherichia coli outer membrane-spanning protein re-incorporated in phospholipid planar bilayers generates aqueous channels similar to those of matrix porin. One phosphoporin trimer contains three pores which are induced simultaneously but fluctuate separately between open and closed states. Membrane potential shifts this two-state equilibrium in favour of closed channels. This negative resistance occurs at lower potentials than with matrix porin channels. The phosphoporin channel is poorly anion selective for small solutes. Polyphosphates and other phosphorylated molecules specifically inhibit phosphoporin pore conductance to small ions, a property which is specific to phosphoporin. There is an excellent correlation between the effect of such solutes measured in planar bilayers and their inhibitory effect on beta-lactam antibiotic uptake in vivo by phosphoporin. It is concluded that the phosphoporin channel contains a selectivity filter which is only efficient for larger molecules, most probably through basic residues.     

Binding of the anticonvulsant drug lamotrigine and the neurotoxin batrachotoxin to voltage-gated sodium channels induces conformational changes associated with block and steady-state activation

Voltage-gated sodium channels are dynamic membrane proteins characterized by rapid conformational changes that switch the molecule between closed resting, activated, and inactivated states. Sodium channels are specifically blocked by the anticonvulsant drug lamotrigine, which preferentially binds to the channel pore in the inactivated open state. Batrachotoxin is a lipid-soluble alkaloid that causes steady-state activation and binds in the inner pore of the sodium channel with overlapping but distinct molecular determinants from those of lamotrigine. Using circular dichroism spectroscopy on purified voltage-gated sodium channels from Electrophorus electricus, the secondary structures associated with the mixture of states present at equilibrium in the absence of these ligands were compared with specific stabilized states in their presence. As the channel shifts to open states, there appears to be a significant change in secondary structure to a more alpha-helical conformation. The observed changes are consistent with increased order involving the S6 segments that form the pore, the domain III-IV linker, and the P-loops that form the outer pore and selectivity filter. A molecular model has been constructed for the sodium channel based on its homology with the pore-forming regions of bacterial potassium channels, and automated docking of the crystal structure of lamotrigine with this model produces a structure in which the close contacts of the drug are with the residues previously identified by mutational studies as forming the binding site for this drug.     

First observation of a beta-turn conformation fused with the oxy-analogue of an alpha-turn: the molecular structure of a model peptide of the C-terminal part of gramicidin A

The molecular structure of N-tert-butyloxycarbonyl-D-leucyl-L-phenylalanyl ethanolamide (t-Boc-D-Leu-L-Phe-EA), a protected analogue of the C-terminal dipeptide of the membrane-active linear antibiotic gramicidin A, has been determined by X-ray diffraction. One of the two independent molecules in the asymmetric unit is characterized by a chain reversal stabilized by an intramolecular, three-centre, double hydrogen bonding. It represents the first experimental evidence for a beta-turn conformation fused with the oxy-analogue of an alpha-turn.     

A Structural and Thermodynamic Analysis of Novatrone and Flavin Mononucleotide Heteroassociation in Aqueous Solution by <Superscript>1</Superscript>H NMR Spectroscopy

A heteroassociation of the antitumor antibiotic novatrone (NOV) and flavin mononucleotide (FMN) in aqueous solution was studied by one- and two-dimentional ¹H NMR spectroscopy (500 MHz) to elucidate the molecular mechanism of the possible combined action of the antibiotic and the vitamin. The equilibrium reaction constants, the induced proton chemical shifts, and the thermodynamic parameters (ΔH and Δ_S) of the NOV and FMN heteroassociation were determined from the concentration and temperature dependences of proton chemical shifts of the aromatic molecules. The most favorable structure of the 1 : 1 NOV-FMN complex was determined by both the method of molecular mechanics (X-PLOR software) and the induced proton chemical shifts of the molecules. An analysis of the results suggests that the NOV-FMN intermolecular complexes are mainly stabilized by stacking interactions of their aromatic chromophores. An additional stabilization is possible due to intermolecular hydrogen bonds. It was concluded that the aromatic molecules of vitamins, in particular, FMN, can form energetically favorable heterocomplexes with aromatic antitumor antibiotics in aqueous solutions, which could result in a modulation of their medical and biological action.     

Anthracycline antibiotic arugomycin binds in both grooves of the DNA helix simultaneously: an NMR and molecular modelling study.

Perturbations to the 1H and 31P chemical shifts of DNA resonances together with twenty-four intermolecular nuclear Overhauser effects show that the anthracycline antibiotic arugomycin intercalates between the basepairs of the hexamer duplex d(5'-GCATGC)2 at the 5'-CpA and 5'-TpG binding sites. In the complex two drug molecules are bound per duplex with full retention of the dyad symmetry. Arugomycin adopts a threaded binding orientation with chains of sugars positioned in both the major and minor groove of the helix simultaneously. The complex is stabilized by hydrogen bonding, electrostatic and van der Waals interactions principally in the major groove and involving substituents on the rigidly oriented bicycloamino-glucose sugar of the antibiotic. A specific hydrogen bond is identified between the C2'-hydroxyl and the guanine N7 at the intercalation site. Together, interactions in the major groove appear to account for the intercalation specificity of arugomycin that requires both a guanine and thymine at the intercalation site. We are unable to identify any sequence specific interactions between the minor groove and the arugarose sugar (S1) which binds only weakly, through van der Walls contacts, over the d(GCA).d(TGC) trinucleotide sequence. The data indicate that the sugar chains of arugomycin are flexible and play little part in the interaction of the antibiotic with DNA. The intensity of sequential internucleotide NOEs identifies the intercalation site as being assymmetric. A family of conformers computed using restrained energy minimisation and molecular dynamics indicate that basepair buckling is a feature of the anthracycline intercalation site that may serve to maximise intermolecular van der Waals interactions by wrapping the basepairs around the antibiotic chromophore.     

A structural and thermodynamic analysis of novatrone and flavin mononucleotide heteroassociation in aqueous solution by 1H NMR spectroscopy

A heteroassociation of antitumor antibiotic novatrone (NOV) and flavin mononucleotide (FMN) in aqueous solution was studied by one- and two-dimentional 1H NMR spectroscopy (500 MHz) to elucidate the molecular mechanism of the possible combined action of the antibiotic and vitamin. The equilibrium reaction constants, induced proton chemical shifts, and the thermodynamic parameters (deltaH and deltaS) of the NOV and FMN heteroassociation were determined from the concentration and temperature dependences of proton chemical shifts of the aromatic molecules. The most favorable structure of the 1 : 1 NOV-FMN complex was determined by both the method of molecular mechanics (X-PLOR software) and the induced proton chemical shifts of the molecules. An analysis of the results suggests that the NOV-FMN intermolecular complexes are mainly stabilized by stacking interactions of their aromatic chromophores. An additional stabilization is possible due to intermolecular hydrogen bonds. It was concluded that the aromatic molecules of vitamins, in particular, FMN, can form energetically favorable heterocomplexes with aromatic antitumor antibiotics in aqueous solutions, which could result in a modulation of their medical and biological action.     

Crystal structure of paromomycin docked into the eubacterial ribosomal decoding A site

BACKGROUND: Aminoglycoside antibiotics interfere with translation in both gram-positive and gram-negative bacteria by binding to the tRNA decoding A site of the 16S ribosomal RNA. RESULTS: Crystals of complexes between oligoribonucleotides incorporating the sequence of the ribosomal A site of Escherichia coli and the aminoglycoside paromomycin have been solved at 2.5 A resolution. Each RNA fragment contains two A sites inserted between Watson-Crick pairs. The paromomycin molecules interact in an enlarged deep groove created by two bulging and one unpaired adenines. In both sites, hydroxyl and ammonium side chains of the antibiotic form 13 direct hydrogen bonds to bases and backbone atoms of the A site. In the best-defined site, 8 water molecules mediate 12 other hydrogen bonds between the RNA and the antibiotics. Ring I of paromomycin stacks over base G1491 and forms pseudo-Watson-Crick contacts with A1408. Both the hydroxyl group and one ammonium group of ring II form direct and water-mediated hydrogen bonds to the U1495oU1406 pair. The bulging conformation of the two adenines A1492 and A1493 is stabilized by hydrogen bonds between phosphate oxygens and atoms of rings I and II. The hydrophilic sites of the bulging A1492 and A1493 contact the shallow groove of G=C pairs in a symmetrical complex. CONCLUSIONS: Water molecules participate in the binding specificity by exploiting the antibiotic hydration shell and the typical RNA water hydration patterns. The observed contacts rationalize the protection, mutation, and resistance data. The crystal packing mimics the intermolecular contacts induced by aminoglycoside binding in the ribosome.     

A New Crystal Structure of the Bifunctional Antibiotic Simocyclinone D8 Bound to DNA Gyrase Gives Fresh Insight into the Mechanism of Inhibition

Simocyclinone D8 (SD8) is an antibiotic produced by Streptomyces antibioticus that targets DNA gyrase. A previous structure of SD8 complexed with the N-terminal domain of the DNA gyrase A protein (GyrA) suggested that four SD8 molecules stabilized a tetramer of the protein; subsequent mass spectrometry experiments suggested that a protein dimer with two symmetry-related SD8s was more likely. This work describes the structures of a further truncated form of the GyrA N-terminal domain fragment with and without SD8 bound. The structure with SD8 has the two SD8 molecules bound within the same GyrA dimer. This new structure is entirely consistent with the mutations in GyrA that confer SD8 resistance and, by comparison with a new apo structure of the GyrA N-terminal domain, reveals the likely conformation changes that occur upon SD8 binding and the detailed mechanism of SD8 inhibition of gyrase. Isothermal titration calorimetry experiments are consistent with the crystallography results and further suggest that a previously observed complex between SD8 and GyrB is ~ 1000-fold weaker than the interaction with GyrA.     

Prepore to pore transition of a cholesterol-dependent cytolysin visualized by electron microscopy

Perfringolysin O (PFO), a soluble toxin secreted by the pathogenic Clostridium perfringens, forms large homo-oligomeric pore complexes comprising up to 50 PFO molecules in cholesterol-containing membranes. In this study, electron microscopy (EM) and single-particle image analysis were used to reconstruct two-dimensional (2D) projection maps from images of oligomeric PFO prepore and pore complexes formed on cholesterol-rich lipid layers. The projection maps are characterized by an outer and an inner ring of density peaks. The outer rings of the prepore and pore complexes are very similar; however, the protein densities that make up the inner ring of the pore complex are more intense and discretely resolved than they are for the prepore complex. The change in inner-ring protein density is consistent with a mechanism in which the monomers within the prepore complex make a transition from a partially disordered state to a more ordered transmembrane beta-barrel in the pore complex. Finally, the orientation of the monomers within the oligomeric complexes was determined by visualization of streptavidin (SA) molecules bound to biotinylated cysteine-substituted residues predicted to face either the inner or outer surface of the oligomeric pore complex. This study provides an unprecedented view of the conversion of the PFO prepore to pore complex.     

Interaction between aromatic antibiotics and vitamins: 1H NMR analysis of the heteroassociation of nicotinamide and anthracycline antitumor antibiotics

The interaction between anthracycline antitumor antibiotics daunomycin and novatrone and the vitamin nicotinamide has been investigated by one- and two-dimensional 1H NMR spectroscopy (500 MHz). Due to significant differences in structures of the chromophores of interacting molecules, a two-site heteroassociation model has been developed, allowing the arrangement of one and two nicotinamide molecules on the chromophore of the antibiotic. The equilibrium association constant, thermodynamical parameters (deltaH, deltaS) of the heteroassociation of nicotinamide with daunomycin and novatrone and the induced proton chemical shifts in the heterocomplexes have been determined from the concentration and temperature dependences of proton chemical shifts of interacting molecules. The most favorable structures of 1:1 nicotinamide--daunomycin and nicotinamide-novatrone heteroassociation complexes have been determined using both the molecular mechanics methods (X-PLOR software) and the calculated values of induced proton chemical shifts. Analysis of the results obtained allows one to conclude that two nicotinamide molecules cannot simultaneously bind on one side of the chromophore of antibiotic. Heterocomplexes of the vitamin with the antibiotics with a stoichiometry 1:1 are mainly stabilized by the stacking of aromatic chromophores.     

Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains

The pore-lining amino acids of ion channel proteins reside on the interface between a polar (the pore) and a nonpolar environment (the rest of the protein). The structural dynamics of this region, which physically controls ionic flow, are essential components of channel gating. Using large-conductance, Ca(2+)-dependent K(+) (BK) channels, we devised a systematic charge-substitution method to probe conformational changes in the pore region during channel gating. We identified a deep-pore residue (314 in hSlo1) as a marker of structural dynamics. We manipulated the charge states of this residue by substituting amino acids with different valence and pKa, and by adjusting intracellular pH. We found that the charged states of the 314 residues stabilized an open state of the BK channel. With models based on known structures of related channels, we postulate a dynamic rearrangement of the deep-pore region during BK channel opening/closing, which involves a change of the degree of pore exposure for 314.     

Interaction of the noncovalent molecular adapter, beta-cyclodextrin, with the staphylococcal alpha-hemolysin pore

Cyclodextrins act as noncovalent molecular adapters when lodged in the lumen of the alpha-hemolysin (alphaHL) pore. The adapters act as binding sites for channel blockers, thereby offering a basis for the detection of a variety of organic molecules with alphaHL as a biosensor element. To further such studies, it is important to find conditions under which the dwell time of cyclodextrins in the lumen of the pore is extended. Here, we use single-channel recording to explore the pH- and voltage-dependence of the interaction of beta-cyclodextrin (betaCD) with alphaHL. betaCD can access its binding site only from the trans entrance of pores inserted from the cis side of a bilayer. Analysis of the binding kinetics shows that there is a single binding site for betaCD, with an apparent equilibrium dissociation constant that varies by >100-fold under the conditions explored. The dissociation rate constant for the neutral betaCD molecule varies with pH and voltage, a result that is incompatible with two states of the alphaHL pore, one of high and the other of low affinity. Rather, the data suggest that the actual equilibrium dissociation constant for the alphaHL. betaCD complex varies continuously with the transmembrane potential.