Structural and Dynamical Studies of Humanin in Water and TFE/Water Mixture: A Molecular Dynamics Simulation

Summary

Proline-rich peptides are known to adopt preferentially the extended polyproline II (PPII) helical conformation, which is involved in several protein-protein recognition events. By resorting to molecular modelling techniques, we wished to investigate the extent to which PPII helices could be used for the formation of isohelical peptide-DNA complexes leading to the selective recognition of the major groove of B-DNA. For that purpose, we have grafted to a cationic intercalator, 9-amino-acridine, an oligopeptide having the sequence: Pro-Arg-Pro-Pro-Arg-Pro-Pro-Arg-Pro-Pro-Asp-Pro-Pro. Each residue in the sequence was set in the D configuration, to prevent enzymatic hydrolysis, and each Arg residue was designed to target O6/N7 of a guanine base following the intercalation site. The Asp residue was designed to target a cytosine base, whilst simultaneously forming a bidentate complex with the Arg three residues upstream. Energy-minimization, using the JUMNA procedure, led to the following conclusions: 1) major groove binding is favoured over minor groove or exclusive binding to the phosphates by large energy differences, of over 50 and 90 kcal/mole, respectively; 2) the two best bound sequences are those having three successive guanine bases on the same DNA strand, immediately adjacent to the intercalation site. Sequence d(CGGGC G), encountered in the Primer Binding Site of the HIV retrovirus, thus ranks amongst the best-bound sequences; 3) replacement of an individual guanine amongst the three ones upstream of the intercalation site, by an adenine base, weakens by > 6 kcal/mole the binding energetics; 4) the conformational rigidity of the DNA-bound PPII helix should enable for a modulation of the base sequence selectivity, by appropriate replacements of the Arg and Asp residues. Thus sequence CGGCAAG, also encountered in the HIV genome, could be targeted by an oligopeptide having the sequence Pro-Arg-Pro-Pro-Asp-Pro-Pro- Asn-Pro-Pro-Asn-Pro-Pro-Arg-Ala.     

Heparin Cofactor II,a Serine Protease Inhibitor,Promotes Angiogenesis via Activation of the AMP-activated Protein Kinase-Endothelial Nitric-oxide Synthase Signaling Pathway

We previously clarified that heparin cofactor II (HCII), a serine proteinase inhibitor, exerts various protective actions on cardiovascular diseases in both experimental and clinical studies. In the present study, we aimed to clarify whether HCII participates in the regulation of angiogenesis. Male heterozygous HCII-deficient (HCII^+/−) mice and male littermate wild-type (HCII^+/+) mice at the age of 12–16 weeks were subjected to unilateral hindlimb ligation surgery. Laser speckle blood flow analysis showed that blood flow recovery in response to hindlimb ischemia was delayed in HCII^+/− mice compared with that in HCII^+/+ mice. Capillary number, arteriole number, and endothelial nitric-oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and liver kinase B1 (LKB1) phosphorylation in ischemic muscles were decreased in HCII^+/− mice. Human purified HCII (h-HCII) administration almost restored blood flow recovery, capillary density, and arteriole number as well as phosphorylation levels of eNOS, AMPK, and LKB1 in ischemic muscles of HCII^+/− mice. Although treatment with h-HCII increased phosphorylation levels of eNOS, AMPK, and LKB1 in human aortic endothelial cells (HAECs), the h-HCII-induced eNOS phosphorylation was abolished by compound C, an AMPK inhibitor, and by AMPK siRNA. In a similar fashion, tube formation, proliferation, and migration of HAECs were also promoted by h-HCII treatment and were abrogated by pretreatment with compound C. HCII potentiates the activation of vascular endothelial cells and the promotion of angiogenesis in response to hindlimb ischemia via an AMPK-eNOS signaling pathway. These findings suggest that HCII is a novel therapeutic target for treatment of patients with peripheral circulation insufficiency.     

Elastic Properties of Zinc-blende G a N,A l N and I n N from Molecular Dynamics

Molecular dynamics calculations of the adiabatic elastic constants of group III-Nitrides for temperatures ranging from 300 to 900 K have been performed. The results show good agreement with first-principles calculations. The moduli decreased with increasing temperature. The structural properties of zinc-blende GaN, AlN and InN are reported. Good agreement between the calculated and experimental values of the lattice constant, the cohesion energy, and the bulk modulus and its derivative are obtained.     

A Molecular Dynamics Study of Human Defensins HBD-1 and HNP-3 in Water

Abstract

Mammalian defensins are crucial components of the innate immune system. They are characterized by three disulfide bridges and exhibit broad spectrum antibacterial activity. The spacing between the cysteines and disulfide connectivities in the two classes of defensins, the α- and β-forms, are different. The structural motif of 3 β-strands appears to be conserved in α and β-defensins despite differences in disulfide connectivities and spacing between cysteines. In this study, Molecular Dynamics Simulations (MDS) have been carried out to study the conformational behavior of α- and β-defensins with and without disulfide bridges. Our results indicate that β-strands in the C-terminal region of HBD-1 and HNP-3 do not unfold during the course of MDS. The segment adopting α-helix in HBD-1 unfolds early during the simulations. The backbone hydrogen bonds in HBD-1 and HNP-3 are broken during MDS. When the disulfide bonds are absent, the N-terminal β-strand unfolds by 20 ns but β-strands are observed in the C-terminal region of HNP-3. HBD-1, without disulfide bridges, unfolds to a greater extent during the course of the MDS. Examination of distances between sulfur atoms of cysteines without disulfide bridges during the simulations indicate that there is no specific preference for native disulfide bridges, which could be the reason for the experimental observation of non-native disulfide bridge formation during chemical synthesis of human α- and β-defensins. Since defensins with non-native disulfide bridges are biologically active, the exact three dimensional structures observed for native HBD-1 and HNP-3 does not appear to be essential for exhibiting antibacterial activity.     

Preparation of Extended Metaphase Chromosomes from Human Cultured Cells Using a Topoisomerase II Inhibitor,ICRF-193

Effects of ICRF-193, a topoisomerase II inhibitor, on metaphase chromosome preparations were examined. A short-time exposure of this drug to human HL60 cells in a suspension culture before harvest resulted in obtaining more extended metaphase chromosomes. The length of chromosome 6 identified by fluorescence in situ hybridization was twice as long with this drug treatment. Together with effectiveness for adherent HepG2 cells, these results suggest that treatments with ICRF-193 provide a simple and reliable method for extended metaphase chromosome preparations from cultured cells.     

Conserved Water-mediated Hydrogen Bond Network between TM-I, -II, -VI,and -VII in 7TM Receptor Activation

Five highly conserved polar residues connected by a number of structural water molecules together with two rotamer micro-switches, TrpVI:13 and TyrVII:20, constitute an extended hydrogen bond network between the intracellular segments of TM-I, -II, -VI, and -VII of 7TM receptors. Molecular dynamics simulations showed that, although the fewer water molecules in rhodopsin were relatively movable, the hydrogen bond network of the β2-adrenergic receptor was fully loaded with water molecules that were surprisingly immobilized between the two rotamer switches, both apparently being in their closed conformation. Manipulations of the rotamer state of TyrVII:20 and TrpVI:13 demonstrated that these residues served as gates for the water molecules at the intracellular and extracellular ends of the hydrogen bond network, respectively. TrpVI:13 at the bottom of the main ligand-binding pocket was shown to apparently function as a catching trap for water molecules. Mutational analysis of the β2-adrenergic receptor demonstrated that the highly conserved polar residues of the hydrogen bond network were all important for receptor signaling but served different functions, some dampening constitutive activity (AsnI:18, AspII:10, and AsnVII:13), whereas others (AsnVII:12 and AsnVII:16) located one helical turn apart and sharing a water molecule were shown to be essential for agonist-induced signaling. It is concluded that the conserved water hydrogen bond network of 7TM receptors constitutes an extended allosteric interface between the transmembrane segments being of crucial importance for receptor signaling and that part of the function of the rotamer micro-switches, TyrVII:20 and TrpVI:13, is to gate or trap the water molecules.     

The Structure of Human Apolipoprotein A-IV as Revealed by Stable Isotope-assisted Cross-linking,Molecular Dynamics,and Small Angle X-ray Scattering

Apolipoprotein (apo)A-IV plays important roles in dietary lipid and glucose metabolism, and knowledge of its structure is required to fully understand the molecular basis of these functions. However, typical of the entire class of exchangeable apolipoproteins, its dynamic nature and affinity for lipid has posed challenges to traditional high resolution structural approaches. We previously reported an x-ray crystal structure of a dimeric truncation mutant of apoA-IV, which showed a unique helix-swapping molecular interface. Unfortunately, the structures of the N and C termini that are important for lipid binding were not visualized. To build a more complete model, we used chemical cross-linking to derive distance constraints across the full-length protein. The approach was enhanced with stable isotope labeling to overcome ambiguities in determining molecular span of the cross-links given the remarkable similarities in the monomeric and dimeric apoA-IV structures. Using 51 distance constraints, we created a starting model for full-length monomeric apoA-IV and then subjected it to two modeling approaches: (i) molecular dynamics simulations and (ii) fitting to small angle x-ray scattering data. This resulted in the most detailed models yet for lipid-free monomeric or dimeric apoA-IV. Importantly, these models were of sufficient detail to direct the experimental identification of new functional residues that participate in a “clasp” mechanism to modulate apoA-IV lipid affinity. The isotope-assisted cross-linking approach should prove useful for further study of this family of apolipoproteins in both the lipid-free and -bound states.     

Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47^phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47^phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47^phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47^phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22^phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47^phox−/− coronary microvascular cells. Compared with wild-type p47^phox cDNA transfected cells, the single mutation of S379A completely blocked p47^phox membrane translocation, binding to p22^phox and endothelial O₂^⨪ production in response to acute stimulation of PKC. p47^phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47^phox conformational changes and NADPH oxidase-dependent superoxide production by cells.     

Noncovalent interactions and coordination reactions in the systems consisting of copper(II) ions, aspartic acid and diamines

Interactions of aspartic acid between 1,3-diaminopropane (tn) and 1,4-diaminobutane (Put) in metal-free systems as well as in the systems including copper(II) ions were studied. The composition and overall stability constants of the complexes formed were determined by the potentiometric method. The interaction centres and coordination sites were identified by spectroscopic methods. Each of the ligands has both negative and positive interaction centres. In aspartic acid such centres are carboxyl groups and amine group, while in the polyamine molecules – protonated amine groups. The centres are also the potential sites of the coordination of metal ions. Analysis of the log K_e values of the adducts in the systems with polyamines has shown that the stability of the adducts in the metal-free systems depends on a significant degree on the steric factor that is the length of the polyamine. In some species the inversion effect, hitherto not reported in literature, was found. In the ternary systems including Cu(II) ions, only protonated species are formed, including molecular complexes with intermolecular interactions and metallation through the oxygen atoms of carboxyl groups and amine groups of the amino acid. In the adducts the protonated diamine is in the outer coordination sphere and is involved in noncovalent interactions with the anchoring CuH(Asp) or Cu(Asp) complexes.     

Water in Photosystem II: Structural,functional and mechanistic considerations

Water is clearly important for the functioning of Photosystem II (PSII). Apart from being the very substrate that needs to be transported in this water oxidation enzyme, water is also vital for the transport of protons to and from the catalytic center as well as other important co-factors and key residues in the enzyme. The latest crystal structural data of PSII have enabled detailed analyses of the location and possible function of water molecules in the enzyme. Significant progress has also been made recently in the investigation of channels and pathways through the protein complex. Through these studies, the mechanistic significance of water for PSII is becoming increasingly clear. An overview and discussion of key aspects of the current research on water in PSII is presented here. The role of water in three other systems (aquaporin, bacteriorhodopsin and cytochrome P450) is also outlined to illustrate further points concerning the central significance that water can have, and potential applications of these ideas for continued research on PSII. It is advocated that water be seen as an integral part of the protein and far from a mere solvent.     

Structural dynamics of the cell wall precursor lipid II in the presence and absence of the lantibiotic nisin

Representing a physiological “Achilles' heel”, the cell wall precursor lipid II (L_II) is a prime target for various classes of antibiotics. Over the years L_II-binding agents have been recognized as promising candidates and templates in the search for new antibacterial compounds to complement or replace existing drugs. To elucidate the molecular structural basis underlying L_II functional mechanism and to better understand if and how lantibiotic binding alters the molecular behavior of L_II, we performed molecular dynamics (MD) simulations of phospholipid membrane-embedded L_II in the absence and presence of the L_II-binding lantibiotic nisin. In a series of 2 × 4 independent, unbiased 100 ns MD simulations we sampled the conformational dynamics of nine L_II as well as nine L_II–nisin complexes embedded in an aqueous 150 mM NaCl/POPC phospholipid membrane environment. We found that nisin binding to L_II induces a reduction of L_II mobility and flexibility, an outward shift of the L_II pentapeptide, an inward movement of the L_II disaccharide section, and an overall deeper insertion of the L_II tail group into the membrane. The latter effect might indicate an initial step in adopting a stabilizing, scaffold-like structure in the process of nisin-induced membrane leakage. At the same time nisin conformation and L_II interaction remain similar to the 1WCO L_II–nisin NMR solution structure.     

Role of Water Molecules in Structure and Energetics of Pseudomonas aeruginosa Lectin I Interacting with Disaccharides

Calcium-dependent lectin I from Pseudomonas aeruginosa (PA-IL) binds specifically to oligosaccharides presenting an α-galactose residue at their nonreducing end, such as the disaccharides αGal1–2βGalOMe, αGal1–3βGalOMe, and αGal1–4βGalOMe. This provides a unique model for studying the effect of the glycosidic linkage of the ligands on structure and thermodynamics of the complexes by means of experimental and theoretical tools. The structural features of PA-IL in complex with the three disaccharides were established by docking and molecular dynamics simulations and compared with those observed in available crystal structures, including PA-IL·αGal1–2βGalOMe complex, which was solved at 2.4 Å resolution and reported herein. The role of a structural bridge water molecule in the binding site of PA-IL was also elucidated through molecular dynamics simulations and free energy calculations. This water molecule establishes three very stable hydrogen bonds with O6 of nonreducing galactose, oxygen from Pro-51 main chain, and nitrogen from Gln-53 main chain of the lectin binding site. Binding free energies for PA-IL in complex with the three disaccharides were investigated, and the results were compared with the experimental data determined by titration microcalorimetry. When the bridge water molecule was included in the free energy calculations, the simulations predicted the correct binding affinity trends with the 1–2-linked disaccharide presenting three times stronger affinity ligand than the other two. These results highlight the role of the water molecule in the binding site of PA-IL and indicate that it should be taken into account when designing glycoderivatives active against P. aeruginosa adhesion.     

Computation Confirms Contraction: A Molecular Dynamics Study of Liquid Methanol,Water and a Methanol-Water Mixture

It may be questioned whether potential models that have been developed independently for two different pure compounds would behave properly when used in computer simulations of mixtures of these compounds. Since they are optimized for the pure compounds there is no guarantee whatsoever that the terms describing the interaction between dissimilar molecules are correct. If the simulational and experimental values of several thermodynamical properties of the mixture relative to those of the pure compounds agree closely, however, this strongly indicates that no separate optimizations need be carried out for the mixtures. Here we present the results of isothermal-isobaric Molecular Dynamics simulations of liquid methanol, water and equimolar methanol-water mixtures, using simple point charge models. The potential parameters of the models for the pure liquids had been independently optimized. No adjustment of parameters was made for the mixture, but nonetheless the experimental volume contraction and excess enthalpy upon mixing were reproduced almost perfectly.     

Synthesis, characterization, structure, molecular modeling studies and biological activity of sterically crowded Pt(II) complexes containing bis(imidazole) ligands

The syntheses, structures and biological evaluation of a series of cisplatin-like complexes containing bis(imidazole) derivatives - the so-called Joseph ligands - are described. Their cytotoxicity is discussed in terms of their polar surface area, rate of aquation, and lipophilicity. The X-ray crystal structure of the platinum diiodido derivative of dimethyl 2-(di(1H-imidazol-2-yl)methyl)malonate) is reported and compared to those of related systems. Molecular modeling studies are focused on the hydrogen bonding properties of such systems, and their relevance to antitumor activity.     

Significance of the Conserved Tyr352 and Asp380 Residues in the Catalytic Activity of Bacillus stearothermophilus Aminopeptidase II as Evaluated by Site-directed Mutagenesis

The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.5-fold. Detailed analysis of the kinetic consequences in the mutant proteins revealed that the K (m) values were increased 1.9- to 2.6-fold with respect to wild-type enzyme. Catalytic efficiencies (k (cat)/K (m)) of mutant proteins were between 3.5- and 31-fold lower than the corresponding value of the wild-type enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results indicate that residues Tyr352 and Asp380 are essential for the proper function of AP-II.     

Preparation, in vitro screening and molecular modelling of symmetrical bis-quinolinium cholinesterase inhibitors--implications for early myasthenia gravis treatment

This paper describes the preparation and in vitro evaluation of 18 newly prepared bis-quinolinium inhibitors on human recombinant acetylcholinesterase (AChE) and human plasmatic butyrylcholinesterase (BChE). Their inhibitory (IC₅₀) and was compared to the chosen standards ambenonium dichloride, edrophonium chloride, BW284c51 and ethopropazine hydrochloride. One novel compound was found to be a promising inhibitor of hAChE (in nM range) and was better than edrophonium chloride or BW284c51, but was worse than ambenonium chloride. This compound also showed selectivity towards hAChE and it was confirmed as a non-competitive inhibitor of hAChE by kinetic analysis. A molecular modelling study further confirmed its binding to the peripheral active site of hAChE via apparent π-π or π-cationic interactions.     

Conserved Allosteric Hot Spots in the Transmembrane Domains of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels and Multidrug Resistance Protein (MRP) Pumps

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.     

Design,synthesis and biological research of novel N-phenylbenzamide-4-methylamine acridine derivatives as potential topoisomerase I/II and apoptosis-inducing agents

A series of novel N-phenylbenzamide-4-methylamine acridine derivatives were designed and synthesized based initially on the structure of amsacrine (m-AMSA). Molecular docking suggested that the representative compound 9a had affinity for binding DNA topoisomerase (Topo) II, which was comparable with that of m-AMSA, and furthermore that 9a could have preferential interactions with Topo I. After synthesis of 9a and analogues 9b-9f, these were all tested in vitro and the synthesized compounds displayed potent antiproliferative activity against three different cancer cell lines (K562, CCRF-CEM and U937). Among them, compounds 9b, 9c and 9d exhibiting the highest activity with IC₅₀ value ranging from 0.82 to 0.91 μM against CCRF-CEM cells. In addition, 9b and 9d also showed high antiproliferative activity against U937 cells, with IC₅₀ values of 0.33 and 0.23 μM, respectively. The pharmacological mechanistic studies of these compounds were evaluated by Topo I/II inhibition, western blot assay and cell apoptosis detection. In summary, 9b effectively inhibited the activity of Topo I/II and induced DNA damage in CCRF-CEM cells and, moreover, significantly induced cell apoptosis in a concentration-dependent manner. These observations provide new information and guidance for the structural optimization of more novel acridine derivatives.     

Power, precaution, Type II error and sampling design in assessment of environmental impacts

Increasingly, environmental managers attempt to incorporate precautionary principles into decision making. In any quantitative analysis of impacts, precaution is closely related to the power of the analysis to detect an impact. Designs of sampling to detect impacts are, however, complex because of natural spatial and temporal variability and the intrinsic nature of the statistical interactions which define impacts. Here, pulse and press responses and impacts that affect time courses (temporal variance) were modelled to determine the influences of increasing temporal replication—sampling more times in each of several longer periods before and again after an impact.Increasing the number of control or reference locations and number of replicate sample units at each time and place of sampling investigated the influence of spatial replication on power. From numerous scenarios of impacts, with or without natural spatial and temporal interactions (i.e. not caused by an impact), general recommendations are possible. Detecting press impacts requires maximal numbers of control locations. Shorter-term pulse impacts are best detected when the number of periods sampled is maximized. Impacts causing changes in temporal variance are most likely to be detected by sampling with the greatest possible number of periods or times within periods.To allow precautionary decision making, the type of predicted impact should be specified with its magnitude and duration. Only then can sampling be designed to be powerful, thereby allowing precautionary concepts to be invoked.