scholarly journals Interactions of Visinin-like Proteins with Phospho-inositides

2010 ◽  
Vol 63 (3) ◽  
pp. 350 ◽  
Author(s):  
Karl-Heinz Braunewell ◽  
Blessy Paul ◽  
Wassim Altarche-Xifro ◽  
Cornelia Noack ◽  
Kristian Lange ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Electrostatic Interactions ◽  
Specific Binding ◽  
Surface Membrane ◽  
Living Cells ◽  
Head Group ◽  
Subcellular Membrane ◽  
Monolayer Adsorption ◽  
Lysine Residues ◽  
Phosphatidylinositol Phosphates

The subcellular membrane localization of neuronal calcium sensor (NCS) proteins in living cells, such as Visinin-like Proteins-1 (VILIP-1) and VILIP-3, differs substantially. We have followed the hypothesis that the differential localization may be due to the specific binding capabilities of individual VILIPs for phosphatidylinositol phosphates (PIPs). Several highly conserved lysine residues in the N-terminal region could provide favourable electrostatic interactions. Molecular modelling results support a binding site for phospho-inositides in the N-terminal area of VILIP-1, and the involvement of the conserved N-terminal lysine residues in binding the phospho-inositol head group. Experimentally, the binding of VILIP-1 to inositol derivatives was tested by a PIP strip assay, which showed the requirement of phosphorylation of the inositol group for the interaction of the protein with PIPs. Monolayer adsorption measurements showed a preference of VILIP-1 binding to PI(4,5)P2 over PI(3,4,5)P3. The co-localization of VILIP-1 with PI(4,5)P2 at the cell surface membrane in hippocampal neurons further supports the idea of direct interactions of VILIP-1 with PIPs in living cells.

Studies of the Mechanism for Enhanced Cell Surface Factor VIIa/Tissue Factor Activation of Factor X on Fibroblast Monolayers after Their Exposure to N-Ethylmaleimide

1994 ◽  
Vol 72 (06) ◽  
pp. 848-855 ◽  
Author(s):  
Dzung The Le ◽  
Samuel I Rapaport ◽  
L Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Factor Viia ◽  
Cell Damage ◽  
Specific Binding ◽  
Surface Membrane ◽  
Annexin V ◽  
Factor X ◽  
Binding Studies ◽  
Anionic Phospholipid

SummaryFibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

scholarly journals How Insertion of a Single Tryptophan in the N-Terminus of a Cecropin A-Melittin Hybrid Peptide Changes Its Antimicrobial and Biophysical Profile

Membranes ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 48
Author(s):  
Ana Rita Ferreira ◽  
Cátia Teixeira ◽  
Carla F. Sousa ◽  
Lucinda J. Bessa ◽  
Paula Gomes ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Bacterial Infections ◽  
Electron System ◽  
Bacterial Membranes ◽  
Highly Active ◽  
Large Unilamellar Vesicles ◽  
Biophysical Profile ◽  
N Terminus ◽  
Lysine Residues ◽  
Preferential Location

In the era of antibiotic resistance, there is an urgent need for efficient antibiotic therapies to fight bacterial infections. Cationic antimicrobial peptides (CAMP) are promising lead compounds given their membrane-targeted mechanism of action, and high affinity towards the anionic composition of bacterial membranes. We present a new CAMP, W-BP100, derived from the highly active BP100, holding an additional tryptophan at the N-terminus. W-BP100 showed a broader antibacterial activity, demonstrating a potent activity against Gram-positive strains. Revealing a high partition constant towards anionic over zwitterionic large unilamellar vesicles and inducing membrane saturation at a high peptide/lipid ratio, W-BP100 has a preferential location for hydrophobic environments. Contrary to BP100, almost no aggregation of anionic vesicles is observed around saturation conditions and at higher concentrations no aggregation is observed. With these results, it is possible to state that with the incorporation of a single tryptophan to the N-terminus, a highly active peptide was obtained due to the π–electron system of tryptophan, resulting in negatively charged clouds, that participate in cation–π interactions with lysine residues. Furthermore, we propose that W-BP100 action can be achieved by electrostatic interactions followed by peptide translocation.

scholarly journals Kv2.1 cell surface clusters are insertion platforms for ion channel delivery to the plasma membrane

2012 ◽  
Vol 23 (15) ◽  
pp. 2917-2929 ◽  
Author(s):  
Emily Deutsch ◽  
Aubrey V. Weigel ◽  
Elizabeth J. Akin ◽  
Phil Fox ◽  
Gentry Hansen ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Ion Channel ◽  
Protein Trafficking ◽  
Hippocampal Neurons ◽  
Surface Membrane ◽  
Cell Types ◽  
Homogeneous Surface ◽  
Hek Cells ◽  
Membrane Protein Trafficking

Voltage-gated K+ (Kv) channels regulate membrane potential in many cell types. Although the channel surface density and location must be well controlled, little is known about Kv channel delivery and retrieval on the cell surface. The Kv2.1 channel localizes to micron-sized clusters in neurons and transfected human embryonic kidney (HEK) cells, where it is nonconducting. Because Kv2.1 is postulated to be involved in soluble N-ethylmaleimide–sensitive factor attachment protein receptor–mediated membrane fusion, we examined the hypothesis that these surface clusters are specialized platforms involved in membrane protein trafficking. Total internal reflection–based fluorescence recovery after photobleaching studies and quantum dot imaging of single Kv2.1 channels revealed that Kv2.1-containing vesicles deliver cargo at the Kv2.1 surface clusters in both transfected HEK cells and hippocampal neurons. More than 85% of cytoplasmic and recycling Kv2.1 channels was delivered to the cell surface at the cluster perimeter in both cell types. At least 85% of recycling Kv1.4, which, unlike Kv2.1, has a homogeneous surface distribution, is also delivered here. Actin depolymerization resulted in Kv2.1 exocytosis at cluster-free surface membrane. These results indicate that one nonconducting function of Kv2.1 is to form microdomains involved in membrane protein trafficking. This study is the first to identify stable cell surface platforms involved in ion channel trafficking.

scholarly journals A Complete Assessment of Dopamine Receptor-Ligand Interactions through Computational Methods

2019 ◽  
Author(s):  
Beatriz Bueschbell ◽  
Carlos A.V. Barreto ◽  
Antonio J. Preto ◽  
Anke C. Schiedel ◽  
Irina S. Moreira
Keyword(s):  
Dopamine Receptor ◽  
Electrostatic Interactions ◽  
Specific Binding ◽  
Binding Pocket ◽  
Binding Mode ◽  
Receptor Subtypes ◽  
Receptor Ligand ◽  
Pi Stacking ◽  
Ligand Interactions ◽  
New Treatments

Background: Selectively targeting dopamine receptors has been a persistent challenge in the last years for the development of new treatments to combat the large variety of diseases evolving these receptors. Although, several drugs have been successfully brought to market, the subtype-specific binding mode on a molecular basis has not been fully elucidated. Methods: Homology modeling and molecular dynamics were applied to construct robust conformational models of all dopamine receptor subtypes (D1-like and D2-like receptors). Fifteen structurally diverse ligands were docked to these models. Contacts at the binding pocket were fully described in order to reveal new structural findings responsible for DR sub-type specificity. Results: We showed that the number of conformations for a receptor:ligand complex was associated to unspecific interactions > 2.5 Å and hydrophobic contacts, while the decoys binding energy was influenced by specific electrostatic interactions. Known residues such as 3.32Asp, the serine microdomain and the aromatic microdomain were found interacting in a variety of modes (HB, SB, π-stacking). Purposed TM2-TM3-TM7 microdomain was found to form a hydrophobic network involving Orthosteric Binding Pocket (OBP) and Secondary Binding Pocket (SBP). T-stacking interactions revealed as especially relevant for some large ligands such as apomorphine, risperidone or aripiprazole. Conclusions: This in silico approach was successful in showing known receptor-ligand interactions as well as in determining unique combinations of interactions, key for the design of more specific ligands.

scholarly journals The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro

2000 ◽  
Vol 267 (12) ◽  
pp. 3461-3468 ◽  
Author(s):  
Xiao-Song Gong ◽  
Jiang Qi Wen ◽  
Nicholas E. Fisher ◽  
Simon Young ◽  
Christopher J. Howe ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Cytochrome F ◽  
Lysine Residues

Characterization of the exposed carbohydrates on the surface membrane of adult Schistosoma mansoni by analysis of lectin binding

Parasitology ◽  
1980 ◽  
Vol 81 (1) ◽  
pp. 1-15 ◽  
Author(s):  
A. J. G. Simpson ◽  
S. R. Smithers
Keyword(s):  
Schistosoma Mansoni ◽  
Adult Male ◽  
Wheat Germ ◽  
Ricinus Communis ◽  
Specific Binding ◽  
Lectin Binding ◽  
Surface Membrane ◽  
Soybean Agglutinin ◽  
Peanut Agglutinin

SUMMARYThe surface architecture of adult male Schistosoma mansoni was explored using a range of lectins with differing carbohydrate specificities. Highest specific binding was achieved with concanavalin A and the agglutinin of molecular weight 60000 from Ricinus communis; the binding of wheat germ agglutinin was mostly non-specific. Small amounts of peanut agglutinin and soybean agglutinin binding were observed and the binding of these lectins was increased by pre-treating the parasite with neuraminidase. The fucose binding protein of Lotus tetragonolobus failed to bind. These results indicate that mannose and/or glucose, galactose, N-acetyl-glucosamine, N-acetyl-galactosamine and sialic acid are exposed on the surface of the adult male schistosome.

scholarly journals The Tomato Defensin TPP3 Binds Phosphatidylinositol (4,5)-Bisphosphate via a Conserved Dimeric Cationic Grip Conformation To Mediate Cell Lysis

2015 ◽  
Vol 35 (11) ◽  
pp. 1964-1978 ◽  
Author(s):  
Amy A. Baxter ◽  
Viviane Richter ◽  
Fung T. Lay ◽  
Ivan K. H. Poon ◽  
Christopher G. Adda ◽  
...  
Keyword(s):  
Lipid Binding ◽  
Cytolytic Activity ◽  
Membrane Permeabilization ◽  
Site Directed Mutagenesis ◽  
Microbial Pathogens ◽  
Head Group ◽  
Innate Defense ◽  
Plant Defensins ◽  
Oligomeric Complex ◽  
Phosphatidylinositol Phosphates

Defensins are a class of ubiquitously expressed cationic antimicrobial peptides (CAPs) that play an important role in innate defense. Plant defensins are active against a broad range of microbial pathogens and act via multiple mechanisms, including cell membrane permeabilization. The cytolytic activity of defensins has been proposed to involve interaction with specific lipid components in the target cell wall or membrane and defensin oligomerization. Indeed, the defensinNicotiana alatadefensin 1 (NaD1) binds to a broad range of membrane phosphatidylinositol phosphates and forms an oligomeric complex with phosphatidylinositol (4,5)-bisphosphate (PIP2) that facilitates membrane lysis of both mammalian tumor and fungal cells. Here, we report that the tomato defensin TPP3 has a unique lipid binding profile that is specific for PIP2 with which it forms an oligomeric complex that is critical for cytolytic activity. Structural characterization of TPP3 by X-ray crystallography and site-directed mutagenesis demonstrated that it forms a dimer in a “cationic grip” conformation that specifically accommodates the head group of PIP2 to mediate cooperative higher-order oligomerization and subsequent membrane permeabilization. These findings suggest that certain plant defensins are innate immune receptors for phospholipids and adopt conserved dimeric configurations to mediate PIP2 binding and membrane permeabilization. This mechanism of innate defense may be conserved across defensins from different species.

scholarly journals Divalent Ions and the Surface Potential of Charged Phospholipid Membranes

1971 ◽  
Vol 58 (6) ◽  
pp. 667-687 ◽  
Author(s):  
S. G. A. McLaughlin ◽  
G. Szabo ◽  
G. Eisenman
Keyword(s):  
Surface Potential ◽  
Alkaline Earth ◽  
Specific Binding ◽  
Fold Increase ◽  
Phospholipid Bilayer ◽  
Head Group ◽  
Polar Head Group ◽  
Diffuse Double Layer ◽  
Monovalent Ions ◽  
Alkaline Earth Cations

Phospholipid bilayer membranes were bathed in a decimolar solution of monovalent ions, and the conductance produced by neutral carriers of these monovalent cations and anions was used to assess the electric potential at the surface of the membrane. When the bilayers were formed from a neutral lipid, phosphatidylethanolamine, the addition of alkaline earth cations produced no detectable surface potential, indicating that little or no binding occurs to the polar head group with these ions. When the bilayers were formed from a negatively charged lipid, phosphatidylserine, the addition of Sr and Ba decreased the magnitude of the surface potential as predicted by the theory of the diffuse double layer. In particular, the potential decreased 27 mv for a 10-fold increase in concentration in the millimolar-decimolar range. A 10-fold increase in the Ca or Mg concentration also produced a 27 mv decrease in potential in this region, which was again due to screening, but it was necessary to invoke some specific binding to account for the observation that these cations were effective at a lower concentration than Ba or Sr. It is suggested that the ability of the alkaline earth cations to shift the conductance-voltage curves of a nerve along the voltage axis by 20–26 mv for a 10-fold increase in concentration may be due to essentially a screening rather than a binding phenomenon.

scholarly journals The interaction of methanol dehydrogenase and cytochrome cL in the acidophilic methylotroph Acetobacter methanolicus

1991 ◽  
Vol 280 (1) ◽  
pp. 139-146 ◽  
Author(s):  
H T C Chan ◽  
C Anthony
Keyword(s):  
Electrostatic Interactions ◽  
High Ionic Strength ◽  
Methanol Dehydrogenase ◽  
Beta Subunit ◽  
Ionic Interactions ◽  
Oxidoreductase Activity ◽  
Strength Of Interaction ◽  
Lysine Residues ◽  
Arginine Residues ◽  
Cytochrome Cl

The quinoprotein methanol dehydrogenase (MDH) of Acetobacter methanolicus has an alpha 2 beta 2 structure. By contrast with other MDHs, the beta-subunit (approx. 8.5 kDa) does not contain the five lysine residues previously proposed to be involved in ionic interactions with the electron acceptor cytochrome cL. That electrostatic interactions are involved was confirmed by the demonstration that methanol:cytochrome cL oxidoreductase activity was inhibited by high ionic strength (I), the strength of interaction being inversely related to the square root of I. Specific modifiers of arginine residues on MDH inhibited this reaction but not the dye-linked MDH activity. Modification of lysine residues on MDH that altered its charge had no effect on the dye-linked activity but inhibited reaction with cytochrome cL. When the charge was retained on modification of lysine residues, little effect on either activity was observed. Cross-linking experiments confirmed that lysine residues on the alpha-subunit, but not the beta-subunit, are involved in the ‘docking’ process between the proteins.

scholarly journals Evidence for Two CRIB Domains in Phospholipase D2 (PLD2) That the Enzyme Uses to Specifically Bind to the Small GTPase Rac2

2011 ◽  
Vol 286 (18) ◽  
pp. 16308-16320 ◽  
Author(s):  
Hong-Juan Peng ◽  
Karen M. Henkels ◽  
Madhu Mahankali ◽  
Mary C. Dinauer ◽  
Julian Gomez-Cambronero
Keyword(s):  
Specific Binding ◽  
Strong Association ◽  
Living Cells ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Deletion Mutants ◽  
Ph Domain ◽  
Phospholipase D2

Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the Rac2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-Rac2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to Rac2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named “CRIB-1” and “CRIB-2” in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with Rac2 and diminish the FRET signal in living cells. The PLD2-Rac2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent Kd of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to Rac2-GTP than to Rac2-GDP or to a GDP-constitutive Rac2-N17 mutant. Increasing concentrations of recombinant Rac2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, Rac2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects Rac2 in an initial positive feedback, but as Rac2-GTP accumulates in the cell, this constitutes a “termination signal” leading to PLD2 inactivation.

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