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Cell Research Dec 2020In Gram-negative bacteria, phospholipids are major components of the inner membrane and the inner leaflet of the outer membrane, playing an essential role in forming the...
In Gram-negative bacteria, phospholipids are major components of the inner membrane and the inner leaflet of the outer membrane, playing an essential role in forming the unique dual-membrane barrier to exclude the entry of most antibiotics. Understanding the mechanisms of phospholipid translocation between the inner and outer membrane represents one of the major challenges surrounding bacterial phospholipid homeostasis. The conserved MlaFEDB complex in the inner membrane functions as an ABC transporter to drive the translocation of phospholipids between the inner membrane and the periplasmic protein MlaC. However, the mechanism of phospholipid translocation remains elusive. Here we determined three cryo-EM structures of MlaFEDB from Escherichia coli in its nucleotide-free and ATP-bound conformations, and performed extensive functional studies to verify and extend our findings from structural analyses. Our work reveals unique structural features of the entire MlaFEDB complex, six well-resolved phospholipids in three distinct cavities, and large-scale conformational changes upon ATP binding. Together, these findings define the cycle of structural rearrangement of MlaFEDB in action, and suggest that MlaFEDB uses an extrusion mechanism to extract and release phospholipids through the central translocation cavity.
Topics: Adenosine Triphosphate; Cryoelectron Microscopy; Escherichia coli; Escherichia coli Proteins; Models, Biological; Models, Molecular; Phospholipids; Protein Binding; Protein Structure, Secondary; Protein Subunits
PubMed: 32884137
DOI: 10.1038/s41422-020-00404-6 -
Journal of Dairy Science Feb 2021In this study dairy phospholipid (PL) gels were made using 3 different concentrations of PL (15%, 30%, and 45%) and soybean oil to determine the gel-forming ability and...
In this study dairy phospholipid (PL) gels were made using 3 different concentrations of PL (15%, 30%, and 45%) and soybean oil to determine the gel-forming ability and functional traits that dairy PL have. After 24 h of storage the visual stability, crystal morphology, solid fat content, melting behavior, viscosity, and oil binding capacity of the gels were evaluated. All samples showed visual stability, whereas polarized light microscopy showed that high concentrations of PL reduced PL mobility, preventing tubular micelles from forming at high concentrations of PL (45%). Solid fat content increased with an increase in PL concentration. The melting enthalpy increased as the concentration of PL increased. The viscosity was assessed at 0.01, 0.1, and 1.0 1/s shear rates. A significant difference was observed between the 45% PL samples and the other samples at low and intermediate shear, but at high shear levels, a significant difference was only seen between the 15% PL sample and the other samples. The oil binding capacity showed a significant difference between the 45% PL sample and the other 2 samples. This study shows that dairy PL can be added to a vegetable oil to produce semi-solid material with appropriate functional properties.
Topics: Animals; Chemical Phenomena; Crystallization; Dairy Products; Fats; Gels; Phospholipids; Soybean Oil; Thermodynamics; Viscosity
PubMed: 33189284
DOI: 10.3168/jds.2020-18737 -
Biochimica Et Biophysica Acta.... Sep 2020NMR is a sophisticated method for investigation of structure and dynamics of lipid and protein molecules in membranes. Vibrational spectroscopy is also powerful because... (Review)
Review
NMR is a sophisticated method for investigation of structure and dynamics of lipid and protein molecules in membranes. Vibrational spectroscopy is also powerful because of relatively high resolution and sensitivity, and easier access than NMR. A combined use of these spectroscopies could provide important insights into the membrane biophysics. A structural analysis of phosphatidylethanolamine (PE) bilayers in built-up films by infrared dichroism suggested that polar groups oriented parallel to the membrane surface. A Raman analysis of phosphatidylcholine (PC) revealed that the gauche conformation was preferred for the choline backbone not only in solid, but also in the gel and liquid-crystalline states. The polar group structure of DPPC bilayers in the liquid-crystalline state was determined by analyzing deuterium quadrupole splitting of the choline group and phosphorus chemical shift anisotropy of the phosphate group in combination with restriction of the gauche conformation of the choline group determined by Raman spectroscopy. This was an excellent complementarity of NMR and vibrational spectroscopies. The deuterium quadrupole splitting values mentioned above were found to change on addition of ions such as NaCl, CaCl, and LaCl, suggesting that a structural change takes place on ion binding and the polar group of PC works as an electric charge sensor of membranes. The ion-bound structure was determined by NMR using the restriction from Raman spectroscopy. The PN vector of phosphorylcholine group was inclined by 63° from the membrane surface, while the inclination was 18° in the ion-free form. The deuterium quadrupole splitting values and phosphorus powder patterns revealed that on mixing with phosphatidylglycerol (PG) or cardiolipin (CL), PC did not change its dynamic structure of the glycerol backbone, but PE did. The mixture of PE with PG or CL shared a new dynamic structure, suggesting their adaptive miscibility in the molecular level. PC was molecularly immiscible with any of PE, PG, and CL. The molecular miscibility would regulate not only interactions of proteins with mixed bilayers but also formation of asymmetric lipid membranes. Interactions of crown-ether (CE) modified artificial microbial peptides with phospholipid bilayers were investigated by NMR and FTIR. CE-modified 14-mers with one or two basic amino acid residues revealed position-specific selectivity for the suppression of calcein leakage from PC vesicles but did not for that from PG vesicles, suggesting that structures of the lipid polar groups play crucial roles in different responses of the vesicles to the positively charged peptides. Manipulation of the peptide-polar group interaction can be used for drug design.
Topics: Cardiolipins; Lipid Bilayers; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylethanolamines; Phosphatidylglycerols; Spectrum Analysis, Raman
PubMed: 32407775
DOI: 10.1016/j.bbamem.2020.183352 -
The Journal of Biological Chemistry Dec 2002Syndecan-4 is a transmembrane heparan sulfate proteoglycan that can regulate cell-matrix interactions and is enriched in focal adhesions. Its cytoplasmic domain contains...
Syndecan-4 is a transmembrane heparan sulfate proteoglycan that can regulate cell-matrix interactions and is enriched in focal adhesions. Its cytoplasmic domain contains a central region unlike that of any other vertebrate or invertebrate syndecan core protein with a cationic motif that binds inositol phospholipids. In turn, lipid binding stabilizes the syndecan in oligomeric form, with subsequent binding and activation of protein kinase C. The specificity of phospholipid binding and its potential regulation are investigated here. Highest affinity of the syndecan-4 cytoplasmic domain was seen with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5P)(2)) and phosphatidylinositol 4-phosphate, and both promoted syndecan-4 oligomerization. Affinity was much reduced for 3-phosphorylated inositides while no binding of diacylglycerol was detected. Syndecan-2 cytoplasmic domain had negligible affinity for any lipid examined. Inositol hexakisphosphate, but not inositol tetrakisphosphate, also had high affinity for the syndecan-4 cytoplasmic domain and could compete effectively with PtdIns(4,5)P(2). Since inositol hexaphosphate binding to syndecan-4 does not promote oligomer formation, it is a potential down-regulator of syndecan-4 signaling. Similarly, phosphorylation of serine 183 in syndecan-4 cytoplasmic domain reduced PtdIns(4,5)P(2) binding affinity by over 100-fold, although interaction could still be detected by nuclear magnetic resonance spectroscopy. Only protein kinase Calpha was up-regulated in activity by the combination of syndecan-4 and PtdIns(4,5)P(2), with all other isoforms tested showing minimal response. This is consistent with the codistribution of syndecan-4 with the alpha isoform of protein kinase C in focal adhesions.
Topics: Amino Acid Sequence; Animals; Binding, Competitive; Cell Adhesion; Cells, Cultured; Cytoplasm; Dose-Response Relationship, Drug; Enzyme Activation; Gene Expression Regulation; Humans; Inositol; Isoenzymes; Lipid Metabolism; Magnetic Resonance Spectroscopy; Membrane Glycoproteins; Models, Chemical; Molecular Sequence Data; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phospholipids; Phosphorylation; Protein Binding; Protein Isoforms; Protein Kinase C; Protein Kinase C-alpha; Protein Structure, Tertiary; Proteoglycans; Rats; Serine; Signal Transduction; Syndecan-4; Up-Regulation
PubMed: 12377772
DOI: 10.1074/jbc.M209679200 -
Biochimica Et Biophysica Acta Jun 2007A central principle of signal transduction is the appropriate control of the process so that relevant signals can be detected with fine spatial and temporal resolution.... (Review)
Review
A central principle of signal transduction is the appropriate control of the process so that relevant signals can be detected with fine spatial and temporal resolution. In the case of lipid-mediated signaling, organization and metabolism of specific lipid mediators is an important aspect of such control. Herein, we review the emerging evidence regarding the roles of Sec14-like phosphatidylinositol transfer proteins (PITPs) in the action of intracellular signaling networks; particularly as these relate to membrane trafficking. Finally, we explore developing ideas regarding how Sec14-like PITPs execute biological function. As Sec14-like proteins define a protein superfamily with diverse lipid (or lipophile) binding capabilities, it is likely these under-investigated proteins will be ultimately demonstrated as a ubiquitously important set of biological regulators whose functions influence a large territory in the signaling landscape of eukaryotic cells.
Topics: Animals; Biological Transport; Carrier Proteins; Crystallization; Humans; Membrane Lipids; Metabolic Networks and Pathways; Models, Molecular; Phosphatidylcholines; Phosphatidylinositols; Phospholipid Transfer Proteins; Phospholipids; Protein Binding; Saccharomyces cerevisiae Proteins; Signal Transduction
PubMed: 17512778
DOI: 10.1016/j.bbalip.2007.04.002 -
The Journal of Biological Chemistry Jun 2009The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the...
The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (XPC), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31P NMR methods to estimate internal correlation times (tauc) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 XPC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased tauc. PI-PLC activity and phospholipid tauc are constant between 0.1 and 0.5 XPC. Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC tauc, a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high XPC, kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins.
Topics: Bacillus thuringiensis; Enzyme Activation; Lipid Bilayers; Models, Biological; Phosphatidylcholines; Phosphoinositide Phospholipase C; Phospholipids; Protein Binding; Spectrometry, Fluorescence; Substrate Specificity
PubMed: 19336401
DOI: 10.1074/jbc.M809600200 -
International Journal of Molecular... Apr 2020Toll-like receptor 3 (TLR3) provides the host with antiviral defense by initiating an immune signaling cascade for the production of type I interferons. The X-ray...
Toll-like receptor 3 (TLR3) provides the host with antiviral defense by initiating an immune signaling cascade for the production of type I interferons. The X-ray structures of isolated TLR3 ectodomain (ECD) and transmembrane (TM) domains have been reported; however, the structure of a membrane-solvated, full-length receptor remains elusive. We investigated an all-residue TLR3 model embedded inside a phospholipid bilayer using molecular dynamics simulations. The TLR3-ECD exhibited a ~30°-35° tilt on the membrane due to the electrostatic interaction between the N-terminal subdomain and phospholipid headgroups. Although the movement of dsRNA did not affect the dimer integrity of TLR3, its sugar-phosphate backbone was slightly distorted with the orientation of the ECD. TM helices exhibited a noticeable tilt and curvature but maintained a consistent crossing angle, avoiding the hydrophobic mismatch with the bilayer. Residues from the αD helix and the CD and DE loops of the Toll/interleukin-1 receptor (TIR) domains were partially absorbed into the lower leaflet of the bilayer. We found that the previously unknown TLR3-TIR dimerization interface could be stabilized by the reciprocal contact between αC and αD helices of one subunit and the αC helix and the BB loop of the other. Overall, the present study can be helpful to understand the signaling-competent form of TLR3 in physiological environments.
Topics: Humans; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Phospholipids; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; RNA, Double-Stranded; Structure-Activity Relationship; Toll-Like Receptor 3
PubMed: 32325904
DOI: 10.3390/ijms21082857 -
General Physiology and Biophysics Mar 2004Binding of the tricyclic antidepressant imipramine (IMI) to neutral and negatively charged lipid membranes was investigated using a radioligand binding assay combined... (Comparative Study)
Comparative Study
Binding of the tricyclic antidepressant imipramine (IMI) to neutral and negatively charged lipid membranes was investigated using a radioligand binding assay combined with centrifugation or filtration. Lipid bilayers were composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS). IMI binding isotherms were measured up to IMI concentration of 0.5 mmol/l. Due to electrostatic attraction, binding between the positively charged IMI and the negatively charged surfaces of PS membranes was augmented compared to binding to neutral PC membranes. After correction for electrostatic effects by means of the Gouy-Chapman theory, the binding isotherms were described both by surface partition coefficients and by binding parameters (association constants and binding capacities). It was confirmed that binding of IMI to model membranes is strongly affected by negatively charged phospholipids and that the binding is heterogeneous; in fact, weak surface adsorption and incorporation of the drug into the hydrophobic core of lipid bilayer can be seen and characterized. These results support the hypothesis suggesting that the lipid part of biological membranes plays a role in the mechanism of antidepressant action.
Topics: Animals; Antidepressive Agents, Tricyclic; Binding Sites; Brain; Cattle; Centrifugation; Filtration; Imipramine; Kinetics; Lipid Bilayers; Liposomes; Macromolecular Substances; Membranes, Artificial; Models, Chemical; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Radioligand Assay
PubMed: 15270130
DOI: No ID Found -
Nature Communications May 2021As a large family of membrane proteins crucial for bacterial physiology and virulence, the Multiple Peptide Resistance Factors (MprFs) utilize two separate domains to...
As a large family of membrane proteins crucial for bacterial physiology and virulence, the Multiple Peptide Resistance Factors (MprFs) utilize two separate domains to synthesize and translocate aminoacyl phospholipids to the outer leaflets of bacterial membranes. The function of MprFs enables Staphylococcus aureus and other pathogenic bacteria to acquire resistance to daptomycin and cationic antimicrobial peptides. Here we present cryo-electron microscopy structures of MprF homodimer from Rhizobium tropici (RtMprF) at two different states in complex with lysyl-phosphatidylglycerol (LysPG). RtMprF contains a membrane-embedded lipid-flippase domain with two deep cavities opening toward the inner and outer leaflets of the membrane respectively. Intriguingly, a hook-shaped LysPG molecule is trapped inside the inner cavity with its head group bent toward the outer cavity which hosts a second phospholipid-binding site. Moreover, RtMprF exhibits multiple conformational states with the synthase domain adopting distinct positions relative to the flippase domain. Our results provide a detailed framework for understanding the mechanisms of MprF-mediated modification and translocation of phospholipids.
Topics: Bacterial Proteins; Binding Sites; Biological Transport; Cell Membrane; Cryoelectron Microscopy; Lysine; Membrane Proteins; Models, Molecular; Phosphatidylglycerols; Phospholipids; Protein Binding; Protein Conformation; Protein Multimerization; Recombinant Proteins; Rhizobium tropici
PubMed: 34006869
DOI: 10.1038/s41467-021-23248-z -
Methods in Molecular Biology (Clifton,... 2016Acidic phospholipids are minor membrane lipids but critically important for signaling events. The main acidic phospholipids are phosphatidylinositol phosphates (PIPs... (Review)
Review
Acidic phospholipids are minor membrane lipids but critically important for signaling events. The main acidic phospholipids are phosphatidylinositol phosphates (PIPs also known as phosphoinositides), phosphatidylserine (PS), and phosphatidic acid (PA). Acidic phospholipids are precursors of second messengers of key signaling cascades or are second messengers themselves. They regulate the localization and activation of many proteins, and are involved in virtually all membrane trafficking events. As such, it is crucial to understand the subcellular localization and dynamics of each of these lipids within the cell. Over the years, several techniques have emerged in either fixed or live cells to analyze the subcellular localization and dynamics of acidic phospholipids. In this chapter, we review one of them: the use of genetically encoded biosensors that are based on the expression of specific lipid binding domains (LBDs) fused to fluorescent proteins. We discuss how to design such sensors, including the criteria for selecting the lipid binding domains of interest and to validate them. We also emphasize the care that must be taken during data analysis as well as the main limitations and advantages of this approach.
Topics: Biological Transport; Biosensing Techniques; Molecular Imaging; Molecular Probes; Phospholipids; Protein Binding; Protein Interaction Domains and Motifs
PubMed: 26552684
DOI: 10.1007/978-1-4939-3170-5_15