Charge Clusters Research Articles

Bovine lactoferrin peptidic fragments involved in inhibition of Echovirus 6 in vitro infection

Bovine lactoferrin is a multifunctional glycoprotein folded in two symmetric globular lobes (N- and C-lobes), each being able to bind one ferric ion. We have previously demonstrated that this protein is able to prevent echovirus-induced apoptosis. In the present study, we have investigated both the role of tryptic fragments of bovine lactoferrin and the mechanism of lactoferrin effect on echovirus infection. Results obtained showed that bovine lactoferrin inhibits echovirus-induced cytopathic effect and antigen synthesis in a dose-dependent manner and that this protein is able to prevent viral replication when added not only during the entire cycle of infection but also before, during or after the viral adsorption step. The N-terminal cationic peptide was sufficient to prevent viral binding. Our data suggest that lactoferrin inhibition of echovirus attachment to cell receptors could be mediated by the cluster of positive charges at its N-terminus (lactoferricin).

Molecular Dynamics Simulation of Folding of a Short Helical Peptide with Many Charged Residues

A molecular dynamics simulation of the folding of conantokin-T (con-T), a short helical peptide with 5 helical turns of 21 amino acids with 10 charged residues, was carried out to examine folding pathways for this peptide and to predict the folding rate. In the 18 trajectories run at 300 K, 16 trajectories folded, with an averaged folding time of approximately 50 ns. Two trajectories did not fold in up to 200 ns simulation. The folded structure in folded trajectories is in good agreement with experimental structure. An analysis of the trajectories showed that, at the beginning of a few nanoseconds, helix formation started from residues 5-9 with assistance of a hydrophobic clustering involving Tyr5, Met8, and Leu9. The peptide formed a U-shape mainly due to charge-charge interactions between charged residues at the N- and C-terminus segments. In the next approximately 10 ns, several nonnative charge-charge interactions were broken and nonnative Gla10-Lys18 (this denotes a salt bridge between Gal10 and Lys18) and/or Gla10-Lys19 interactions appeared more frequently in this folding step and the peptide became a fishhook J-shape. From this structure, the peptide folded to the folded state in 7 of all 16 folded trajectories in approximately 15 ns. Alternatively, in approximately 30 ns, the con-T went to a conformation in an L-shape with 4 helical turns and a kink at the Arg13 and Gla14 segment in the other 9 trajectories. Con-T in the L-shape then required another approximately 15 ns to fold into the folded state. In addition, in overall folding times, the former 7 trajectories folded faster with the total folding times all shorter than 45 ns, while the latter 9 trajectories folded at a time longer than 45 ns, resulting in an average folding time of approximately 50 ns. Two major folding intermediates found in 2 nonfolded trajectories are stabilized by charge clusters of 5 and 6 charged residues, respectively. With inclusion of friction and solvent-solvent interactions, which were ignored in the present GB/SA solvation model, the folding time obtained above should be multiplied by a factor of 1.25-1.7 according to a previous, similar simulation study. This results in a folding time of 65-105 ns, slightly shorter than the folding time of 127 ns for an alanine-based peptide of the same length. This suggests that the energy barrier of folding for this type of peptides with many charged residues is slightly lower than alanine-based helical peptides by less than 1 kcal/mol.

Structures and Electronic States of Gadolinium Oxide Clusters

We have carried out ab initio quantum chemical calculations for gadolinium oxide clusters and clarified the most suitable combination of a theoretical method, basis sets, and effective core potentials for large systems of gadolinium oxides. We confirmed the reliability of our calculation by computing the electronic state of GdO molecule whose bond length and binding energy are well known. In order to examine how the electronic states depend on the size of clusters, we calculated several cluster series: GdOn clusters (n=2–4) with negative net charge, (GdO)n colinear and zigzag chain clusters (n=1–4), (GdO)n square pillar clusters (n=4,6,8), and large clusters based on the Gd2O3 cubic crystal (Gd4O1824-·8Q3+ and Gd4O1926-·8Q3+), where Q denotes the point charge. New images of electronic stresses in the clusters can be visualized in terms of the electronic stress tensor density based on the regional density functional theory.

Rearrangements in Thyroid Hormone Receptor Charge Clusters That StabilizeBound 3,5′,5-Triiodo-L-thyronine and Inhibit HomodimerFormation

In this study, we investigated how thyroid hormone (3,5',5-triiodo-l-thyronine, T3) inhibits binding of thyroid hormone receptor (TR) homodimers, but not TR-retinoid X receptor heterodimers, to thyroid hormone response elements. Specifically we asked why a small subset of TRbeta mutations that arise in resistance to thyroid hormone syndrome inhibit both T3 binding and formation of TRbeta homodimers on thyroid hormone response elements. We reasoned that these mutations may affect structural elements involved in the coupling of T3 binding to inhibition of TR DNA binding activity. Analysis of TR x-ray structures revealed that each of these resistance to thyroid hormone syndrome mutations affects a cluster of charged amino acids with potential for ionic bond formation between oppositely charged partners. Two clusters (1 and 2) are adjacent to the dimer surface at the junction of helices 10 and 11. Targeted mutagenesis of residues in Cluster 1 (Arg338, Lys342, Asp351, and Asp355) and Cluster 2 (Arg429, Arg383, and Glu311) confirmed that the clusters are required for stable T3 binding and for optimal TR homodimer formation on DNA but also revealed that different arrangements of charged residues are needed for these effects. We propose that the charge clusters are homodimer-specific extensions of the dimer surface and further that T3 binding promotes specific rearrangements of these surfaces that simultaneously block homodimer formation on DNA and stabilize the bound hormone. Our data yield insight into the way that T3 regulates TR DNA binding activity and also highlight hitherto unsuspected T3-dependent conformational changes in the receptor ligand binding domain.

Effect of a cluster structure on the interaction of electron and magnetic subsystems in LaCa(Sr)MnO epilayers

The structure, electrical, and magnetic properties of epitaxial LaCa(Sr)MnO single crystal films with a clustered structure have been studied. In films with a “metallic” phase content C m 0 ≤0.15, the electric conductivity is determined by the spin-dependent tunneling of charge carriers between “ metallic” clusters, and the magnetoresistance is maximum at T = 4.2 K. The correlated motion of carriers over the system of tunneling-linked clusters leads to the formation of a window in the Coulomb blockade. The interactions between atomic, magnetic, and electron subsystems increase in the vicinity of the dielectric-metal percolation transition (T = 200–210 K), where the metal phase content C m in the samples with C m 0 ≥0.2 reaches a maximum (C m crit = 0.5) due to an increase in the cluster size upon cooling. In this case, the magnetoresistance exhibits a maximum at T = 260 K, on the dielectric side of the percolation transition. Due to the presence of space charge regions at the periphery of the clusters, the content of a ferromagnetic phase is 1.5–2 times that of the “metallic” phase. For this reason, the calculations are performed using a model combining the tunneling conductivity mechanism with the percolation approximation for the description of magnetization. Allowance for the Coulomb interaction between charge carriers and clusters improves the agreement of theory and experiment.

Monopole content of topological clusters: Have Kraan-van Baal calorons been found?

Using smearing of equilibrium lattice fields generated at finite temperature in the confined phase of SU(2) lattice gauge theory, we have investigated the emerging topological objects (clusters of topological charge). Analysing their monopole content according to the Polyakov gauge and the maximally Abelian gauge, we characterize part of them to correspond to nonstatic calorons or static dyons in the context of Kraan-van Baal caloron solutions with non-trivial holonomy. The behaviour of the Polyakov loop inside these clusters and the (model-dependent) topological charges of these objects support this interpretation.

Neuroblastoma cell-adapted yellow fever virus: mutagenesis of the E protein locus involved in persistent infection and its effects on virus penetration and spread

Persistent infection of mouse neuroblastoma NB41A3 cells with yellow fever 17D virus generates viral variants which exhibit defective cell penetration, poor cell-to-cell spread, small plaque size and reduced growth efficiency, caused by substitution of glycine for aspartic acid or glutamic acid at positions 360 and 362 in the envelope protein. These positions occur within a charge cluster, Asp360-Asp361-Glu362, located in domain III, near its interface with domain I. To characterize further the molecular basis for the variant phenotype, a series of mutant viruses containing substitutions at position 360, 361 and 362, were studied for effects on the cell culture properties typical of the neuroblastoma-adapted variant. Most substitutions at position 360 gave rise to viruses that were very defective in cell penetration, growth efficiency and cell-to-cell spread, whereas substitution with glutamic acid yielded a virus indistinguishable from parental yellow fever 17D. Substitution with lysine was not tolerated and substitution with asparagine resulted in frequent wild-type revertants. A glycine residue was not tolerated at position 361, but substitution at 362 yielded a small plaque virus, similar to the effect of substitution at position 360. These data indicate that the yellow fever virus E protein contains a locus within domain III where a negative-charge cluster is important for optimal function of this domain in virus-cell interactions beyond the stage of virus attachment. Modelling predictions suggest that the mutations alter the local properties of the loop within domain III, and may compromise interactions of this domain with an adjacent region of domain I during conformational changes that occur in the E protein in association with virus entry.

Structural Requirements for Rapid Inactivation and Voltage Dependence in Splice Variants of Lobster Shaker Potassium Channels

We studied the properties of currents generated in Xenopus oocytes by nine splice variants of the spiny lobster Shaker gene. These isoforms differ in their amino termini and in the P-loop region of the pore. Both the voltage dependence and kinetic properties of the currents varied significantly, depending on which amino terminus was present. A cluster of net positive charges at the N-terminus was not necessary for rapid inactivation: negatively charged N-termini also inactivated rapidly. There was no obvious correlation between N-terminus length and inactivation rate. These N-terminal effects were additive with a separate set of voltage and kinetic properties controlled by the two alternative P-loop exons.

Critical Regions for Assembly of Vertebrate Nonmuscle Myosin II

Myosin II molecules assemble and form filaments through their C-terminal rod region, and the dynamic filament assembly-disassembly process of nonmuscle myosin II molecules is important for cellular activities. To estimate the critical region for filament formation of vertebrate nonmuscle myosin II, we assessed the solubility of a series of truncated recombinant rod fragments of nonmuscle myosin IIB at various concentrations of NaCl. A C-terminal 248-residue rod fragment (Asp 1729-Glu 1976) was shown by its solubility behavior to retain native assembly features, and two regions within it were found to be necessary for assembly: 35 amino acid residues from Asp 1729 to Thr 1763 and 39 amino acid residues from Ala 1875 to Ala 1913, the latter containing a sequence similar to the assembly competence domain (ACD) of skeletal muscle myosin. Fragments lacking either of the two regions were soluble at any NaCl concentration. We referred to these two regions as nonmuscle myosin ACD1 (nACD1) and nACD2, respectively. In addition, we constructed an alpha-helical coiled-coil model of the rod fragment, and found that a remarkable negative charge cluster (termed N1) and a positive charge cluster (termed P2) were present within nACD1 and nACD2, respectively, besides another positive charge cluster (termed P1) in the amino-terminal vicinity of nACD2. From these results, we propose two major electrostatic interactions that are essential for filament formation of nonmuscle myosin II: the antiparallel interaction between P2 and N1 which is essential for the nucleation step and the parallel interaction between P1 and N1 which is important for the elongation step.

Acidic-rich region of amyloid precursor protein induces glial cell apoptosis

Amyloid precursor protein (APP) has several caspase cleavage sites in its C-terminal cytoplasmic domain and N-terminal extracellular domain. Caspase cleavages of APP at its cytosolic tail may result in releasing the domain and inducing cell death. During apoptosis, the N-terminal domain may also be processed at amino acids 197 and 219 by caspases leading to unmasking of an acidic-rich region (AR). In this study, AR-exposing APP was shown to inhibit cell growth after transfection into RBA-1 astrocytes and BV-2 microglial cells. The recombinant AR from residue 220 to 288 of APP (APP220-288) was produced and its biological activities were analyzed. APP220-288 induced morphological changes, cell death, and DNA fragmentation in BV-2 and RBA-1 cells. However, AR was determined to have no apparent effects in suspension cells, erythroleukemia K562 cells, and Jurkat T cells. The cytotoxicity was depending on negative charge cluster and the apoptotic activity of AR was attributed to the inhibition of cell adhesion. In BV-2 microglial cells, AR significantly stimulated Fas expression, although expressions of the pro-inflammatory cytokine genes were not detected. APP220-288 also induced nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. These findings indicate that the acidic-rich domain of APP may have apoptotic activity due to inhibition of cell adhesion and induction of iNOS and Fas expressions. Moreover, unmasking the apoptosis-induced AR may activate and exacerbate glial cells which in turn lead to further progression of the death program.

Crystal Structure of the Human Lamin A Coil 2B Dimer: Implications for the Head-to-tail Association of Nuclear Lamins

Nuclear intermediate filaments (IFs) are made from fibrous proteins termed lamins that assemble, in association with several transmembrane proteins of the inner nuclear membrane and an unknown number of chromatin proteins, into a filamentous scaffold called the nuclear lamina. In man, three types of lamins with significant sequence identity, i.e. lamin A/C, lamin B1 and B2, are expressed. The molecular characteristics of the filaments they form and the details of the assembly mechanism are still largely unknown. Here we report the crystal structure of the coiled-coil dimer from the second half of coil 2 from human lamin A at 2.2A resolution. Comparison to the recently solved structure of the homologous segment of human vimentin reveals a similar overall structure but a different distribution of charged residues and a different pattern of intra- and interhelical salt bridges. These features may explain, at least in part, the differences observed between the lamin and vimentin assembly pathways. Employing a modeled lamin A coil 1A dimer, we propose that the head-to-tail association of two lamin dimers involves strong electrostatic attractions of distinct clusters of negative charge located on the opposite ends of the rod domain with arginine clusters in the head domain and the first segment of the tail domain. Moreover, lamin A mutations, including several in coil 2B, have been associated with human laminopathies. Based on our data most of these mutations are unlikely to alter the structure of the dimer but may affect essential molecular interactions occurring in later stages of filament assembly and lamina formation.

Structure transition in myosin association with the change of concentration: solubility equilibrium under specified KCl and pH condition.

We observed, for the first time, the elementary process for the ordered self-assembly formation of myosin in solution. It was realized exclusively under the specific condition of 200 mM KCl, 5 mM phosphate buffer, pH 7.08, at 15-20 degrees C, which is called the transition-generating condition (TGC). Described more in detail: pure myosin extracted from rabbit skeletal muscle exhibited the structural transition in its association form only when the myosin concentration c was changed under TGC. The myosin solubility was saturated in both edges of the total myosin concentration c > 10.0 mg/mL (solubility region II) and c < or = 0.25 mg/mL (solubility region I). In the intermediate region, the association structure of myosin changed stepwise with decreasing c. The steps were classified into four regions: region I (c < or = 0.25 mg/mL), II (0.25 < or = c < or = 0.50 mg/mL), III (0.50 < or = c < or = 5.0 mg/mL), and IV (c > 5.0 mg/mL). In each region except II, the plot of the relative soluble myosin concentration c(aq)/c against c(-1) gave a straight line of different slopes, certifying that myosin constructs self-assemblies by the closed association mechanism and that the self-assembly takes dual structures in each region. In region II, a drastic transition occurred in the self-assembled dual structures. Here, a highly associated (insoluble) giant assembly would break into soluble assemblies composed of several myosin molecules. The solubility region I originates a driving force for this structural transition. The basic binding unit of the self-assembly would be a parallel myosin-dimer constructed by the intermolecular axial staggers of 14.3 and 43 nm, as is observed by X-ray diffraction for the thick filament assembly or light meromyosin paracrystals. Myosin could take a single rod-like chain form only in an extremely low concentration region of c < or = c(aq,0) (= 0.053 mg/mL). The association behavior revealed in the present study suggests strongly that the complementary charge cluster and its electrostatic interaction between parallel myosin rods play a crucial role for the ordered self-assembly formation and that the specific electrostatic atmosphere of the solution under TGC is essential to the association mechanism in skeletal muscle myosin, or the thick filament formation of the mammals.

P1-189 Mechanism of AB induced cytotoxicity: toxic AB aggregate associates with cell membrane via positive charge clusters

P1-189 Mechanism of AB induced cytotoxicity: toxic AB aggregate associates with cell membrane via positive charge clusters

D88A mutant of cytochrome P450nor provides kinetic evidence for direct complex formation with electron donor NADH.

The haem-distal pocket of nitric oxide reductase cytochrome P450 contains many Arg and Lys residues that are clustered to form a putative access channel for NADH. Asp88 is the sole negatively charged amino acid in this positive charge cluster, and thus it would be interesting to know its functional role. Here we found the intriguing phenomenon that mutation at this site of P450nor (D88A or D88V) considerably decreased the overall nitric oxide reductase activity without blocking the reducing half reaction in which the ferric enzyme-NO complex is reduced with NADH to yield a specific intermediate (I). The results indicate that the catalytic turnover subsequent to the I formation was blocked by such mutation. This property of the mutants made it possible to perform kinetic analysis of the reduction step, which is impossible with the wild-type P450nor. These results are the first kinetic evidence for direct complex formation between P450nor and an electron donor (NADH or NADPH). The kinetic analysis also showed that the inhibition by chloride ions (Cl(-)) is competitive with respect to NAD(P)H, which highlights the importance of the binding site for Cl(-) (the anion hole) in the interaction with NAD(P)H. We also characterized another mutant (D393A) of P450nor. The results demonstrated that both Asp residues play important roles in the interaction with NADH, whereas the role of Asp88 is unique in that it must be essential for the release of NAD(+) rather than binding to NADH.

Molecular dynamics simulations of two-dimensional clusters of charges

We study structural and spectral properties of finite classical systems of N two-dimensional charged particles, confined by a parabolic potential ∝ r n , and interacting via inverse power-law potentials ∝ 1/r n . Molecular dynamics simulations are performed for different cluster sizes (N =3 0to 230 )a ndn, nvalues 1, 2, 3 and 10. We also analyze the phase transition from a ring-like configuration to a Wigner structure as a function of param- eter nand anisotropy. We compare our results with Monte Carlo simulations of Bedanov and Peeters, obtaining good agreement. In addition, we determine the Voronoi structure of the cluster. Our work complements that of Candido, Rino and Studart, who analyzed confinement in a screened parabolic potential.

Freezing of triangulations

Zero temperature dynamics of two dimensional triangulations of a torus with curvature energy is described. Numerical simulations strongly suggest that the model get frozen in metastable states, made of topological defects on flat surfaces, that group into clusters of same topological charge. It is conjectured that freezing is related to high temperature structure of baby universes.

Interaction Codes within the Family of Mammalian Phox and Bem1p Domain-containing Proteins

The Phox and Bem1p (PB1) domain constitutes a recently recognized protein-protein interaction domain found in the atypical protein kinase C (aPKC) isoenzymes, lambda/iota- and zeta PKC; members of mitogen-activated protein kinase (MAPK) modules like MEK5, MEKK2, and MEKK3; and in several scaffold proteins involved in cellular signaling. Among the last group, p62 and Par6 (partitioning-defective 6) are involved in coupling the aPKCs to signaling pathways involved in cell survival, growth control, and cell polarity. By mutation analyses and molecular modeling, we have identified critical residues at the interaction surfaces of the PB1 domains of aPKCs and p62. A basic charge cluster interacts with an acidic loop and helix both in p62 oligomerization and in the aPKC-p62 interaction. Subsequently, we determined the abilities of mammalian PB1 domain proteins to form heteromeric and homomeric complexes mediated by this domain. We report several novel interactions within this family. An interaction between the cell polarity scaffold protein Par6 and MEK5 was found. Furthermore, p62 interacts both with MEK5 and NBR1 in addition to the aPKCs. Evidence for involvement of p62 in MEK5-ERK5 signaling is presented.

Biochemistry and genetics of mannan-binding lectin (MBL).

Mannose- or mannan-binding lectin (MBL) is a member of the collectin protein family, which includes lung surfactant proteins SP-A and SP-D. Each member consists of similar or identical polypeptide chains with a region of collagen-like sequence followed by a C-type lectin domain. The polypeptides associate in threes to form a subunit containing a collagen-like helix, with three clustered lectin domains. These subunits associate into larger structures, usually with 12-18 polypeptides. The collectins bind to patterns of neutral sugars on surfaces (e.g. of micro-organisms) and mediate effector functions associated with killing/phagocytosis. MBL is the only collectin which activates complement. It resembles in quaternary structure the complement protein C1q, which recognizes targets via charge clusters. Binding of MBL to a surface activates MBL-associated serine proteases (MASPs) attached to MBL, and MASP-2 activates complement proteins C4 and C2. The MASPs are homologous to the C1q-associated proteases, C1r and C1s. MBL therefore activates complement by a mechanism very similar to C1q, and engages the opsonic activity of complement to clear micro-organisms. The serum concentration of MBL is very variable in humans. The variability is largely associated with mutations leading to amino acid substitutions in the collagen-like region which decrease MBL assembly and stability. Many studies demonstrate that MBL deficiency is associated with susceptibility to a range of infectious and inflammatory diseases.

A fast multipole method combined with a reaction field for long-range electrostatics in molecular dynamics simulations: The effects of truncation on the properties of water

We present a combination of the structure adapted multipole method with a reaction field (RF) correction for the efficient evaluation of electrostatic interactions in molecular dynamics simulations under periodic boundary conditions. The algorithm switches from an explicit electrostatics evaluation to a continuum description at the maximal distance that is consistent with the minimum image convention, and, thus, avoids the use of a periodic electrostatic potential. A physically motivated switching function enables charge clusters interacting with a given charge to smoothly move into the solvent continuum by passing through the spherical dielectric boundary surrounding this charge. This transition is complete as soon as the cluster has reached the so-called truncation radius Rc. The algorithm is used to examine the dependence of thermodynamic properties and correlation functions on Rc in the three point transferable intermolecular potential water model. Our test simulations on pure liquid water used either the RF correction or a straight cutoff and values of Rc ranging from 14 Å to 40 Å. In the RF setting, the thermodynamic properties and the correlation functions show convergence for Rc increasing towards 40 Å. In the straight cutoff case no such convergence is found. Here, in particular, the dipole–dipole correlation functions become completely artificial. The RF description of the long-range electrostatics is verified by comparison with the results of a particle-mesh Ewald simulation at identical conditions.

Role of positively charged residues in Chlamydomonas reinhardtii ferredoxin-NADP +-reductase

We have previously reported that ferredoxin-NADP-reductase (FNR) from the photosynthetic alga, Chlamydomonas reinhardtii was fully N-trimethylated in vivo on three specific lysines (K83, K89 and K135) located in two sequence insertions comparative to photosynthetic FNRs. This paper deals with the comparison of the biochemical properties of the “native” algal FNR (thus methylated) and the homologous recombinant protein expressed in E. coli, which did not undergo this post-translational modification. Few differences could be observed between the two forms except that their apparent molecular mass was significantly different. This could be indicative that methylation induced changes in the tertiary structure of FNR, making the protein more compact. Another difference was the fivefold higher Km value for NADPH exhibited by the recombinant protein. However, the binding of ferredoxin (Fd) to its reductase seems to be independent of the methylation status. We have also studied four mutated FNRs, in which each Lys was substituted with a hydrophobic amino acid. The results indicate that a cluster of positive charges including K83 and K89 is required for catalytic activity, like in photosynthetic FNRs although the fine interactions are probably different in the algal protein.