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The Journal of Biological Chemistry Mar 2020Eukaryotic cells are compartmentalized to form organelles, whose functions rely on proper phospholipid and protein transport. Here we determined the crystal structure of...
Eukaryotic cells are compartmentalized to form organelles, whose functions rely on proper phospholipid and protein transport. Here we determined the crystal structure of human VAT-1, a cytosolic soluble protein that was suggested to transfer phosphatidylserine, at 2.2 Å resolution. We found that VAT-1 transferred not only phosphatidylserine but also other acidic phospholipids between membranes Structure-based mutational analyses showed the presence of a possible lipid-binding cavity at the interface between the two subdomains, and two tyrosine residues in the flexible loops facilitated phospholipid transfer, likely by functioning as a gate to this lipid-binding cavity. We also found that a basic and hydrophobic loop with two tryptophan residues protruded from the molecule and facilitated binding to the acidic-lipid membranes, thereby achieving efficient phospholipid transfer.
Topics: Binding Sites; Biological Transport; Crystallography, X-Ray; Humans; Liposomes; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Phosphatidylserines; Phospholipids; Protein Domains; Protein Structure, Tertiary; Tryptophan; Vesicular Transport Proteins
PubMed: 32005660
DOI: 10.1074/jbc.RA119.011019 -
Molecular Pharmaceutics Jun 2018Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by...
Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability ( F) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F. The induction of NL did not further increase drug binding but led to altered F due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.
Topics: 3T3 Cells; Adipocytes; Animals; Biological Availability; Cytoplasm; Hydrogen-Ion Concentration; Lysosomes; Mice; Pharmacokinetics; Phospholipids
PubMed: 29709195
DOI: 10.1021/acs.molpharmaceut.8b00064 -
EMBO Reports Jun 2021In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca ion and...
In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER-anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid-like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51-VAPB interaction is mediated by the FFAT-like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM-tethering function.
Topics: Calcium; Endoplasmic Reticulum; Humans; Mitochondria; Mitochondrial Proteins; Phospholipids; Protein Tyrosine Phosphatases
PubMed: 33938112
DOI: 10.15252/embr.202051323 -
Methods in Molecular Biology (Clifton,... 2018Binding of serine protease inhibitors (serpins) to nonprotein ligands such as glycosaminoglycans or phospholipids has been shown to modify their inhibitory activity...
Binding of serine protease inhibitors (serpins) to nonprotein ligands such as glycosaminoglycans or phospholipids has been shown to modify their inhibitory activity and-at least in the case of SERPINA5-to mediate serpin internalization into cells. Also phospholipid functions may be altered when bound to serpins or other proteins.By interacting with phospholipids, serpins might influence a variety of cellular functions. Binding of proteins to phospholipids can be studied by several methods. Here we describe solid-phase assays, in which pure phospholipids are immobilized on nitrocellulose membranes, PVDF membranes, or microtiter plates. Bound proteins are detected with specific antibodies and labeled secondary antibodies. We also describe a method visualizing binding of phospholipids in suspension by non-denaturing polyacrylamide gel electrophoresis (PAGE) followed by Western blotting.
Topics: Animals; Collodion; Electrophoresis, Polyacrylamide Gel; Humans; Membranes, Artificial; Phospholipids; Polyvinyls; Protein Binding; Serpins
PubMed: 30194597
DOI: 10.1007/978-1-4939-8645-3_8 -
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 -
Nature Structural & Molecular Biology Jan 2021
Topics: ATP-Binding Cassette Transporters; Escherichia coli; Phospholipid Transfer Proteins; Phospholipids; Protein Transport; Proteolipids
PubMed: 33361785
DOI: 10.1038/s41594-020-00546-6 -
Journal of Thrombosis and Haemostasis :... Mar 2022Cellular trauma or activation exposes phosphatidylserine (PS) and the substantially more abundant phospholipid, phosphatidylethanolamine (PE), on the outer layer of the...
BACKGROUND
Cellular trauma or activation exposes phosphatidylserine (PS) and the substantially more abundant phospholipid, phosphatidylethanolamine (PE), on the outer layer of the plasma membrane, thereby allowing binding of many blood clotting proteins. We previously proposed the Anything But Choline (ABC) hypothesis to explain how PS and PE synergize to support binding of clotting proteins with gamma-carboxyglutamate (Gla)-rich domains, which posited that each Gla domain binds to a limited number of PS molecules and multiple PE molecules. However, the minimal number of PS molecules required to stably bind a Gla-domain-containing blood clotting protein in the presence of excess PE was unknown.
OBJECTIVE
To test the ABC hypothesis for factor X by determining the threshold binding requirement of PS molecules under conditions of PS-PE synergy.
METHODS
We used surface plasmon resonance to investigate the stoichiometry of factor X binding to nanoscale membrane bilayers (Nanodiscs) of varying phospholipid composition.
RESULTS AND CONCLUSIONS
We quantified 1.05 ± 0.2 PS molecules per bound factor X molecule in Nanodiscs containing a mixture of 10% PS, 60% PE, and 30% phosphatidylcholine. Hence, there appears to be one truly PS-specific binding site per Gla domain, while the remaining membrane binding interactions can be satisfied by PE.
Topics: Binding Sites; Cell Membrane; Factor X; Humans; Phosphatidylserines; Phospholipids; Protein Binding
PubMed: 34894064
DOI: 10.1111/jth.15620 -
The Journal of Biological Chemistry Aug 2022Biological membranes are composed of a wide variety of lipids. Phosphoinositides (PIPns) in the membrane inner leaflet only account for a small percentage of the total...
Biological membranes are composed of a wide variety of lipids. Phosphoinositides (PIPns) in the membrane inner leaflet only account for a small percentage of the total membrane lipids but modulate the functions of various membrane proteins, including ion channels, which play important roles in cell signaling. KcsA, a prototypical K channel that is small, simple, and easy to handle, has been broadly examined regarding its crystallography, in silico molecular analysis, and electrophysiology. It has been reported that KcsA activity is regulated by membrane phospholipids, such as phosphatidylglycerol. However, there has been no quantitative analysis of the correlation between direct lipid binding and the functional modification of KcsA, and it is unknown whether PIPns modulate KcsA function. Here, using contact bubble bilayer recording, we observed that the open probability of KcsA increased significantly (from about 10% to 90%) when the membrane inner leaflet contained only a small percentage of PIPns. In addition, we found an increase in the electrophysiological activity of KcsA correlated with a larger number of negative charges on PIPns. We further analyzed the affinity of the direct interaction between PIPns and KcsA using microscale thermophoresis and observed a strong correlation between direct lipid binding and the functional modification of KcsA. In conclusion, our approach was able to reconstruct the direct modification of KcsA by PIPns, and we propose that it can also be applied to elucidate the mechanism of modification of other ion channels by PIPns.
Topics: Bacterial Proteins; Membrane Lipids; Phosphatidylinositols; Phospholipids; Potassium Channels
PubMed: 35839854
DOI: 10.1016/j.jbc.2022.102257 -
Biological Chemistry Mar 2018Human phospholipid scramblase 3 (hPLSCR3) is a single pass transmembrane protein that plays a vital role in fat metabolism, mitochondrial function, structure,...
Human phospholipid scramblase 3 (hPLSCR3) is a single pass transmembrane protein that plays a vital role in fat metabolism, mitochondrial function, structure, maintenance and apoptosis. The mechanism of action of scramblases remains still unknown, and the role of scramblases in phospholipid translocation is heavily debated. hPLSCR3 is the only member of scramblase family localized to mitochondria and is involved in cardiolipin translocation at the mitochondrial membrane. Direct biochemical evidence of phospholipid translocation by hPLSCR3 is yet to be reported. Functional assay in synthetic proteoliposomes upon Ca2+ and Mg2+ revealed that, apart from cardiolipin, recombinant hPLSCR3 translocates aminophospholipids such as NBD-PE and NBD-PS but not neutral phospholipids. Point mutation in hPLSCR3 (F258V) resulted in decreased Ca2+ binding affinity. Functional assay with F258V-hPLSCR3 led to ~50% loss in scramblase activity in the presence of Ca2+ and Mg2+. Metal ion-induced conformational changes were monitored by intrinsic tryptophan fluorescence, circular dichroism, surface hydrophobicity changes and aggregation studies. Our results revealed that Ca2+ and Mg2+ bind to hPLSCR3 and trigger conformational changes mediated by aggregation. In summary, we suggest that the metal ion-induced conformational change and the aggregation of the protein are essential for the phospholipid translocation by hPLSCR3.
Topics: Binding Sites; Calcium; Humans; Magnesium; Phospholipid Transfer Proteins; Phospholipids; Point Mutation
PubMed: 29337693
DOI: 10.1515/hsz-2017-0309