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.

Permeabilization of Fungal Membranes by Plant Defensins Inhibits Fungal Growth

1999 ◽  
Vol 65 (12) ◽  
pp. 5451-5458 ◽  
Author(s):  
Karin Thevissen ◽  
Franky R. G. Terras ◽  
Willem F. Broekaert
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Antifungal Activity ◽  
Raphanus Sativus ◽  
Plasma Membranes ◽  
Fungal Growth ◽  
Membrane Permeabilization ◽  
Plant Defensin ◽  
Sytox Green ◽  
Plant Defensins ◽  
Fungal Growth Inhibition

ABSTRACT We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 μM), strong permeabilization was detected that could be strongly suppressed by cations in the medium. This permeabilization appears to rely on direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1 μM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N. crassa. Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its antifungal activity. However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistantS. cerevisiae mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins.

scholarly journals Comparison of type 5d autotransporter phospholipases demonstrates a correlation between high activity and intracellular pathogenic lifestyle

2019 ◽  
Vol 476 (18) ◽  
pp. 2657-2676
Author(s):  
Thomas Trunk ◽  
Michael A. Casasanta ◽  
Christopher C. Yoo ◽  
Daniel J. Slade ◽  
Jack C. Leo
Keyword(s):  
Enzymatic Activity ◽  
Lipase Activity ◽  
Bacterial Species ◽  
Lipid Binding ◽  
Human Pathogen ◽  
Secretion Systems ◽  
Animal Pathogens ◽  
Recombinant Type ◽  
Phosphatidylinositol Phosphates ◽  
Pla1 Activity

Abstract Autotransporters, or type 5 secretion systems, are widespread surface proteins of Gram-negative bacteria often associated with virulence functions. Autotransporters consist of an outer membrane β-barrel domain and an exported passenger. In the poorly studied type 5d subclass, the passenger is a patatin-like lipase. The prototype of this secretion pathway is PlpD of Pseudomonas aeruginosa, an opportunistic human pathogen. The PlpD passenger is a homodimer with phospholipase A1 (PLA1) activity. Based on sequencing data, PlpD-like proteins are present in many bacterial species. We characterized the enzymatic activity, specific lipid binding and oligomeric status of PlpD homologs from Aeromonas hydrophila (a fish pathogen), Burkholderia pseudomallei (a human pathogen) and Ralstonia solanacearum (a plant pathogen) and compared these with PlpD. We demonstrate that recombinant type 5d-secreted patatin domains have lipase activity and form dimers or higher-order oligomers. However, dimerization is not necessary for lipase activity; in fact, by making monomeric variants of PlpD, we show that enzymatic activity slightly increases while protein stability decreases. The lipases from the intracellular pathogens A. hydrophila and B. pseudomallei display PLA2 activity in addition to PLA1 activity. Although the type 5d-secreted lipases from the animal pathogens bound to intracellular lipid targets, phosphatidylserine and phosphatidylinositol phosphates, hydrolysis of these lipids could only be observed for FplA of Fusobacterium nucleatum. Yet, we noted a correlation between high lipase activity in type 5d autotransporters and intracellular lifestyle. We hypothesize that type 5d phospholipases are intracellularly active and function in modulation of host cell signaling events.

scholarly journals Synthesis of Highly Water-Soluble Adamantyl Phosphoinositide Derivatives

2015 ◽  
Vol 68 (4) ◽  
pp. 543 ◽  
Author(s):  
Mark Gregory ◽  
Meng-Xin Yin ◽  
Malcolm J. McConville ◽  
Eleanor Williams ◽  
Alex N. Bullock ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Binding Proteins ◽  
Cell Signalling ◽  
Lipid Binding ◽  
Water Soluble ◽  
Side Chains ◽  
Phosphatidylinositol Phosphate ◽  
Phosphatidylinositol Phosphates ◽  
Lipid Binding Proteins ◽  
Natural Lipid

Phosphatidylinositol phosphates are key regulators of cell signalling pathways and membrane trafficking in eukaryotic cells, and there is a need for new chemical probes to further understand how they interact with lipid-binding proteins. Here, the synthesis of phosphatidylinositol phosphate analogues containing adamantyl carboxylic ester groups, in place of the natural lipid side chains, is described. These derivatives are considerably more soluble in water than analogues containing other lipid side chains and do not form large aggregates such as liposomes or micelles. These adamantyl analogues bind to known phosphoinositide-binding proteins with similar affinities to native ligands and will facilitate future studies on the substrate specificities of these proteins involving cocrystallisation studies with proteins.

Interactions of peripheral proteins with model membranes as viewed by molecular dynamics simulations

2014 ◽  
Vol 42 (5) ◽  
pp. 1418-1424 ◽  
Author(s):  
Antreas C. Kalli ◽  
Mark S. P. Sansom
Keyword(s):  
Lipid Bilayers ◽  
Molecular Mechanisms ◽  
Phospholipid Composition ◽  
Md Simulations ◽  
Multiscale Simulation ◽  
Lipid Binding ◽  
Coarse Grained ◽  
Peripheral Proteins ◽  
Dynamics Simulations ◽  
Phosphatidylinositol Phosphates

Many cellular signalling and related events are triggered by the association of peripheral proteins with anionic lipids in the cell membrane (e.g. phosphatidylinositol phosphates or PIPs). This association frequently occurs via lipid-binding modules, e.g. pleckstrin homology (PH), C2 and four-point-one, ezrin, radixin, moesin (FERM) domains, present in peripheral and cytosolic proteins. Multiscale simulation approaches that combine coarse-grained and atomistic MD simulations may now be applied with confidence to investigate the molecular mechanisms of the association of peripheral proteins with model bilayers. Comparisons with experimental data indicate that such simulations can predict specific peripheral protein–lipid interactions. We discuss the application of multiscale MD simulation and related approaches to investigate the association of peripheral proteins which contain PH, C2 or FERM-binding modules with lipid bilayers of differing phospholipid composition, including bilayers containing multiple PIP molecules.

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.

scholarly journals Genetic and Functional Analyses of PptA, a Phospho-Form Transferase Targeting Type IV Pili in Neisseria gonorrhoeae

2007 ◽  
Vol 190 (1) ◽  
pp. 387-400 ◽  
Author(s):  
Cecilia L. Næssan ◽  
Wolfgang Egge-Jacobsen ◽  
Ryan W. Heiniger ◽  
Matthew C. Wolfgang ◽  
Finn Erik Aas ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Metal Binding ◽  
Catalytic Mechanism ◽  
Phylogenetic Analyses ◽  
Site Directed Mutagenesis ◽  
Head Group ◽  
Sequence Alignments ◽  
Type Iv ◽  
Covalent Modifications ◽  
Division Cell

ABSTRACT The PilE pilin subunit protein of Neisseria gonorrhoeae undergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). The pilin phospho-form transferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here that pptA expression is coupled at the level of transcription to its distal gene, murF, in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves a trans-esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE in Pseudomonas aeruginosa resulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique to N. gonorrhoeae.

scholarly journals A structural basis for inhibition of the complement initiator protease C1r by Lyme disease spirochetes

2021 ◽  
Author(s):  
Ryan J. Garrigues ◽  
Alexandra D. Powell ◽  
Michal Hammel ◽  
Jon T. Skare ◽  
Brandon L. Garcia
Keyword(s):  
Lyme Disease ◽  
Molecular Model ◽  
Innate Immune ◽  
Site Directed Mutagenesis ◽  
Microbial Pathogens ◽  
Size Exclusion ◽  
Structural Basis ◽  
Classical Pathway ◽  
Human Pathogens ◽  
Complement Components

AbstractThe complement system is a proteolytic cascade involving dozens of soluble and membrane-associated proteins that function in recognition, opsonization, and elimination of foreign or damaged cells of host origin. Complement evasion is a hallmark of certain human microbial pathogens such as Borreliella burgdorferi, the causative agent of Lyme disease, as well as related spirochetes. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway of complement. Previously we have shown that BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease subcomponent of the first component of complement, C1. To understand the structural basis for BBK32-mediated C1r inhibition, we performed size exclusion chromatography-coupled small angle x-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with a truncated form of activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. Analysis of the structure shows that BBK32 inhibits C1r by occluding important substrate interaction subsites (i.e. S1 and S1’) within the active site of C1r and reveals a surprising role for the C1r B loop in the BBK32 inhibitory model and overall C1r enzyme function. The studies reported here provide a structural basis for classical pathway-specific inhibition by a human pathogen and provide new insight into means for therapeutic inhibition of C1r in complement-related diseases.Significance StatementComplement is a major arm of the innate immune system, which functions in the recognition and destruction of pathogenic microbes. Bloodborne pathogens like the Lyme disease spirochete, Borreliella burgdorferi, have evolved sophisticated mechanisms to evade complement-mediated killing. In this study we provide a structural basis for how a complement inhibitor from B. burgdorferi, designated BBK32, inhibits the initiating protease of the classical pathway of complement, C1r. A molecular understanding for the unique complement inhibitory properties of BBK32 improves our knowledge of how human pathogens combat one arm of the innate immune response and reveals new ways to target C1r in complement-related autoimmune and inflammatory diseases.

scholarly journals Structure and functionality of a multimeric human COQ7:COQ9 complex

2021 ◽  
Author(s):  
Mateusz Manicki ◽  
Halil Aydin ◽  
Luciano A. Abriata ◽  
Katherine A. Overmyer ◽  
Rachel M. Guerra ◽  
...  
Keyword(s):  
Antioxidant Defense ◽  
Binding Capacity ◽  
Coenzyme Q ◽  
Lipid Binding ◽  
Mitochondrial Inner Membrane ◽  
Access Channel ◽  
Redox Active ◽  
Oligomeric Complex ◽  
Dynamics Simulations ◽  
Functional Analyses

Coenzyme Q (CoQ, ubiquinone) is a redox-active lipid essential for core metabolic pathways and antioxidant defense. CoQ is synthesized upon the mitochondrial inner membrane by an ill-defined 'complex Q' metabolon. Here we present a structure and functional analyses of a substrate- and NADH-bound oligomeric complex comprised of two complex Q subunits: the hydroxylase COQ7, which performs the penultimate step in CoQ biosynthesis, and the prenyl lipid-binding protein COQ9. We reveal that COQ7 adopts a modified ferritin-like fold with an extended hydrophobic access channel whose substrate binding capacity is enhanced by COQ9. Using molecular dynamics simulations, we further show that two COQ7:COQ9 heterodimers form a curved tetramer that deforms the membrane, potentially opening a pathway for CoQ intermediates to translocate from within the bilayer to the proteins' lipid-binding sites. Two such tetramers assemble into a soluble octamer, closed like a capsid, with lipids captured within. Together, these observations indicate that COQ7 and COQ9 cooperate to access hydrophobic precursors and coordinate subsequent synthesis steps toward producing mature CoQ.

Sign in / Sign up

Export Citation Format

Share Document