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ACS Chemical Biology Apr 2024Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous...
Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear. Here, we investigate the binding of Ca to Syt-1, first in the absence of lipids, using native mass spectrometry to evaluate individual binding affinities. Syt-1 binds to one Ca with a ∼ 45 μM. Each subsequent binding affinity ( ≥ 2) is successively unfavorable. Given that Syt-1 has been reported to bind anionic phospholipids to modulate the Ca binding affinity, we explored the extent that Ca binding was mediated by selected anionic phospholipid binding. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P) and dioleoylphosphatidylserine (DOPS) positively modulated Ca binding. However, the extent of Syt-1 binding to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P) was reduced with increasing [Ca]. Overall, we find that specific lipids differentially modulate Ca binding. Given that these lipids are enriched in different subcellular compartments and therefore may interact with Syt-1 at different stages of the synaptic vesicle cycle, we propose a regulatory mechanism involving Syt-1, Ca, and anionic phospholipids that may also control some aspects of vesicular exocytosis.
Topics: Calcium; Exocytosis; Neurotransmitter Agents; Phospholipids; Synaptic Transmission; Synaptic Vesicles; Synaptotagmin I; Animals; Rats
PubMed: 38566504
DOI: 10.1021/acschembio.3c00772 -
Nature Communications 2013Inwardly rectifying potassium (Kir) channels regulate multiple tissues. All Kir channels require interaction of phosphatidyl-4,5-bisphosphate (PIP2) at a...
Inwardly rectifying potassium (Kir) channels regulate multiple tissues. All Kir channels require interaction of phosphatidyl-4,5-bisphosphate (PIP2) at a crystallographically identified binding site, but an additional nonspecific secondary anionic phospholipid (PL(-)) is required to generate high PIP2 sensitivity of Kir2 channel gating. The PL(-)-binding site and mechanism are yet to be elucidated. Here we report docking simulations that identify a putative PL(-)-binding site, adjacent to the PIP2-binding site, generated by two lysine residues from neighbouring subunits. When either lysine is mutated to cysteine (K64C and K219C), channel activity is significantly decreased in cells and in reconstituted liposomes. Directly tethering K64C to the membrane by modification with decyl-MTS generates high PIP2 sensitivity in liposomes, even in the complete absence of PL(-)s. The results provide a coherent molecular mechanism whereby PL(-) interaction with a discrete binding site results in a conformational change that stabilizes the high-affinity PIP2 activatory site.
Topics: Anions; Humans; Molecular Docking Simulation; Phospholipids; Potassium Channels, Inwardly Rectifying
PubMed: 24270915
DOI: 10.1038/ncomms3786 -
BioMed Research International 2014On the canalicular membranes of hepatocytes, several ABC transporters are responsible for the secretion of bile lipids. Among them, ABCB4, also called MDR3, is essential... (Review)
Review
On the canalicular membranes of hepatocytes, several ABC transporters are responsible for the secretion of bile lipids. Among them, ABCB4, also called MDR3, is essential for the secretion of phospholipids from hepatocytes into bile. The biliary phospholipids are associated with bile salts and cholesterol in mixed micelles, thereby reducing the detergent activity and cytotoxicity of bile salts and preventing cholesterol crystallization. Mutations in the ABCB4 gene result in progressive familial intrahepatic cholestasis type 3, intrahepatic cholestasis of pregnancy, low-phospholipid-associated cholelithiasis, primary biliary cirrhosis, and cholangiocarcinoma. In vivo and cell culture studies have demonstrated that the secretion of biliary phospholipids depends on both ABCB4 expression and bile salts. In the presence of bile salts, ABCB4 located in nonraft membranes mediates the efflux of phospholipids, preferentially phosphatidylcholine. Despite high homology with ABCB1, ABCB4 expression cannot confer multidrug resistance. This review summarizes our current understanding of ABCB4 functions and physiological relevance, and discusses the molecular mechanism for the ABCB4-mediated efflux of phospholipids.
Topics: ATP Binding Cassette Transporter, Subfamily B; Amino Acid Sequence; Animals; Bile; Bile Acids and Salts; Biological Transport; Humans; Molecular Sequence Data; Pharmaceutical Preparations; Phospholipids
PubMed: 25133187
DOI: 10.1155/2014/954781 -
Biochimica Et Biophysica Acta Sep 2015The molecular activity of Na,K-ATPase and other P2 ATPases like Ca(2+)-ATPase is influenced by the lipid environment via both general (physical) and specific (chemical)... (Review)
Review
The molecular activity of Na,K-ATPase and other P2 ATPases like Ca(2+)-ATPase is influenced by the lipid environment via both general (physical) and specific (chemical) interactions. Whereas the general effects of bilayer structure on membrane protein function are fairly well described and understood, the importance of the specific interactions has only been realized within the last decade due particularly to the growing field of membrane protein crystallization, which has shed new light on the molecular details of specific lipid-protein interactions. It is a remarkable observation that specific lipid-protein interactions seem to be evolutionarily conserved, and conformations of specifically bound lipids at the lipid-protein surface within the membrane are similar in crystal structures determined with different techniques and sources of the protein, despite the rather weak lipid-protein interaction energy. Studies of purified detergent-soluble recombinant αβ or αβFXYD Na,K-ATPase complexes reveal three separate functional effects of phospholipids and cholesterol with characteristic structural selectivity. The observations suggest that these three effects are exerted at separate binding sites for phophatidylserine/cholesterol (stabilizing), polyunsaturated phosphatidylethanolamine (stimulatory), and saturated PC or sphingomyelin/cholesterol (inhibitory), which may be located within three lipid-binding pockets identified in recent crystal structures of Na,K-ATPase. The findings point to a central role of direct and specific interactions of different phospholipids and cholesterol in determining both stability and molecular activity of Na,K-ATPase and possible implications for physiological regulation by membrane lipid composition. This article is part of a special issue titled "Lipid-Protein Interactions."
Topics: Animals; Crystallography, X-Ray; Humans; Membrane Lipids; Membrane Proteins; Models, Molecular; Phospholipids; Protein Binding; Protein Structure, Tertiary; Sodium-Potassium-Exchanging ATPase
PubMed: 25791351
DOI: 10.1016/j.bbamem.2015.03.012 -
Sheng Li Xue Bao : [Acta Physiologica... Dec 2006Phospholipid scramblase 1 (PLSCR1) is a calcium-binding, multiply palmitoylated type II endofacial plasma membrane protein, while unpalmitoylated PLSCR1 protein can...
Phospholipid scramblase 1 (PLSCR1) is a calcium-binding, multiply palmitoylated type II endofacial plasma membrane protein, while unpalmitoylated PLSCR1 protein can import into the nucleus, where it binds to genomic DNA. Although the original work showed that PLSCR1 contributes to the transbilayer movement of phospholipids, the following studies revealed that PLSCR1 expression can be induced by some cytokines such as interferon, epidermal growth factor, and also by leukemic cell differentiation-inducing agents such as all-trans retinoic acid (ATRA) and phorbol 12-myristate 13-acetate (PMA). PLSCR1 was also shown to interact with several protein kinases including c-Abl, c-Src, protein kinase Cdelta as well as some other proteins such as onzin, suggesting the roles of PLSCR1 in cell signaling. Indeed, the current evidence proposes that PLSCR1 contributes to cell proliferation, differentiation, apoptosis, and plays roles in the pathogenesis of cancers, especially leukemia.
Topics: Apoptosis; Cell Differentiation; Cell Proliferation; Cytokines; Humans; Leukemia; Phospholipid Transfer Proteins; Phospholipids; Protein Kinases
PubMed: 17173184
DOI: No ID Found -
Biochimica Et Biophysica Acta.... Feb 2018Heat shock protein 90 (Hsp90) is an essential molecular chaperone with versatile functions in cell homeostatic control under both normal and stress conditions. Hsp90 has...
Heat shock protein 90 (Hsp90) is an essential molecular chaperone with versatile functions in cell homeostatic control under both normal and stress conditions. Hsp90 has been found to be expressed on the cell surface, but the mechanism of Hsp90 association to the membrane remains obscure. In this study, the direct interaction of Hsp90 and phospholipid vesicles was characterized, and the role of Hsp90 on membrane physical state was explored. Using surface plasmon resonance (SPR), we observed a strong interaction between Hsp90 and different compositions of lipid. Hsp90 had a preference to bind with more unsaturated phospholipid species and the affinity was higher with negatively charged lipids than zwitterionic lipids. Increasing the mole fraction of cholesterol in the phospholipid led to a decrease of binding affinity to Hsp90. Circular dichroism (CD) spectroscopy of Hsp90 in PC membranes showed more α-helix structure than in aqueous buffer. The differential scanning calorimeter (DSC) and fluorescence polarization results showed Hsp90 could affect the transition temperature and fluidity of the bilayer. We postulate from these results that the association between Hsp90 and membranes may involve both electrostatic and hydrophobic force, and constitute a possible mechanism that modulates membrane lipid order during thermal fluctuations.
Topics: Animals; Binding, Competitive; Calorimetry, Differential Scanning; Cholesterol; Circular Dichroism; Ducks; Fluorescence Polarization; HSP90 Heat-Shock Proteins; Lipid Bilayers; Membrane Fluidity; Membrane Lipids; Phosphatidylcholines; Phospholipids; Protein Binding; Protein Structure, Secondary; Surface Plasmon Resonance; Transition Temperature
PubMed: 29166573
DOI: 10.1016/j.bbamem.2017.11.011 -
The Journal of Biological Chemistry May 1999Cytosolic phospholipase A2 (cPLA2) plays a key role in the generation of arachidonic acid, a precursor of potent inflammatory mediators. Intact cPLA2 is known to...
Cytosolic phospholipase A2 (cPLA2) plays a key role in the generation of arachidonic acid, a precursor of potent inflammatory mediators. Intact cPLA2 is known to translocate in a calcium-dependent manner from the cytosol to the nuclear envelope and endoplasmic reticulum. We show here that the C2 domain of cPLA2 alone is sufficient for this calcium-dependent translocation in living cells. We have identified sets of exposed hydrophobic residues in loops known as calcium-binding region (CBR) 1 and CBR3, which surround the C2 domain calcium-binding sites, whose mutation dramatically decreased phospholipid binding in vitro without significantly affecting calcium binding. Mutation of a residue that binds calcium ions (D43N) also eliminated phospholipid binding. The same mutations that prevent phospholipid binding of the isolated C2 domain in vitro abolished the calcium-dependent translocation of cPLA2 to internal membranes in vivo, suggesting that the membrane targeting is driven largely by direct interactions with the phospholipid bilayer. Using fluorescence quenching by spin-labeled phospholipids for a series of mutants containing a single tryptophan residue at various positions in the cPLA2 C2 domain, we show that two of the calcium-binding loops, CBR1 and CBR3, penetrate in a calcium-dependent manner into the hydrophobic core of the phospholipid bilayer, establishing an anchor for docking the domain onto the membrane.
Topics: Biological Transport; Calcium; Cytosol; Escherichia coli; Mutation; Phospholipases A; Phospholipases A2; Phospholipids; Protein Binding; Protein Structure, Tertiary
PubMed: 10329700
DOI: 10.1074/jbc.274.21.14979 -
Proceedings of the National Academy of... Mar 2017Membrane protein function can be affected by the physical state of the lipid bilayer and specific lipid-protein interactions. For Na,K-ATPase, bilayer properties can...
Membrane protein function can be affected by the physical state of the lipid bilayer and specific lipid-protein interactions. For Na,K-ATPase, bilayer properties can modulate pump activity, and, as observed in crystal structures, several lipids are bound within the transmembrane domain. Furthermore, Na,K-ATPase activity depends on phosphatidylserine (PS) and cholesterol, which stabilize the protein, and polyunsaturated phosphatidylcholine (PC) or phosphatidylethanolamine (PE), known to stimulate Na,K-ATPase activity. Based on lipid structural specificity and kinetic mechanisms, specific interactions of both PS and PC/PE have been inferred. Nevertheless, specific binding sites have not been identified definitively. We address this question with native mass spectrometry (MS) and site-directed mutagenesis. Native MS shows directly that one molecule each of 18:0/18:1 PS and 18:0/20:4 PC can bind specifically to purified human Na,K-ATPase (αβ). By replacing lysine residues at proposed phospholipid-binding sites with glutamines, the two sites have been identified. Mutations in the cytoplasmic αL8-9 loop destabilize the protein but do not affect Na,K-ATPase activity, whereas mutations in transmembrane helices (TM), αTM2 and αTM4, abolish the stimulation of activity by 18:0/20:4 PC but do not affect stability. When these data are linked to crystal structures, the underlying mechanism of PS and PC/PE effects emerges. PS (and cholesterol) bind between αTM 8, 9, 10, near the FXYD subunit, and maintain topological integrity of the labile C terminus of the α subunit (site A). PC/PE binds between αTM2, 4, 6, and 9 and accelerates the rate-limiting EP-EP conformational transition (site B). We discuss the potential physiological implications.
Topics: Binding Sites; Enzyme Activation; Humans; Mass Spectrometry; Models, Molecular; Molecular Conformation; Phospholipids; Protein Binding; Protein Stability; Sodium-Potassium-Exchanging ATPase
PubMed: 28242691
DOI: 10.1073/pnas.1620799114 -
The Journal of Biological Chemistry Feb 2010Reversible interactions between acidic phospholipids in the cellular membrane and proteins in the cytosol play fundamental roles in a wide variety of physiological...
Reversible interactions between acidic phospholipids in the cellular membrane and proteins in the cytosol play fundamental roles in a wide variety of physiological events. Here, we present a novel approach to the identification of acidic phospholipid-binding proteins using nano-liquid chromatography-tandem mass spectrometry. We found more than 400 proteins, including proteins with previously known acidic phospholipid-binding properties, and confirmed that several candidates, such as Coronin 1A, mDia1 (Diaphanous-related formin-1), PIR121/CYFIP2, EB2 (end plus binding protein-2), KIF21A (kinesin family member 21A), eEF1A1 (translation elongation factor 1alpha1), and TRIM2, directly bind to acidic phospholipids. Among such novel proteins, we provide evidence that Coronin 1A activity, which disassembles Arp2/3-containing actin filament branches, is spatially and temporally regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)). Whereas Coronin 1A co-localizes with PI(4,5)P(2) at the plasma membrane in resting cells, it is dissociated from the plasma membrane during lamellipodia formation where the PI(4,5)P(2) signal is significantly reduced. Our in vitro experiments show that Coronin 1A preferentially binds to PI(4,5)P(2)-containing liposomes and that PI(4,5)P(2) antagonizes the ability of Coronin 1A to disassemble actin filament branches, indicating a spatiotemporal regulation of Coronin 1A via a direct interaction with the plasma membrane lipid. Collectively, our proteomics data provide a list of potential acidic phospholipid-binding protein candidates ranging from the actin regulatory proteins to translational regulators.
Topics: Actin Cytoskeleton; Animals; Brain Chemistry; Cell Membrane; Chromatography, Liquid; Microfilament Proteins; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipids; Protein Binding; Proteins; Proteome; Proteomics; Rats; Tandem Mass Spectrometry
PubMed: 20032464
DOI: 10.1074/jbc.M109.057018 -
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