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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 -
Biochimica Et Biophysica Acta.... Oct 2023Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as... (Review)
Review
Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as several emerging viruses that affect millions of people worldwide. They enter cells by endocytosis, fusing their membrane with the late endosomal one in a pH-dependent manner, so membrane fusion is one of the main targets for obtaining new antiviral inhibitors. The envelope E protein, a class II membrane fusion protein, is responsible for fusion and contains different domains involved in the fusion mechanism, including the fusion peptide. However, other segments, apart from the fusion peptide, have been implicated in the mechanism of membrane fusion, in particular a segment containing a His residue supposed to act as a specific pH sensor. We have used atomistic molecular dynamics to study the binding of the envelope E protein segment containing the conserved His residue in its three different tautomer forms with a complex membrane mimicking the late-endosomal one. We show that this His-containing segment is capable of spontaneous membrane binding, preferentially binds electronegatively charged phospholipids and does not bind cholesterol. Since Flaviviruses have caused epidemics in the past, continue to do so and will undoubtedly continue to do so, this specific segment could characterise a new target that would allow finding effective antiviral molecules against DENV virus in particular and Flaviviruses in general.
Topics: Humans; Viral Envelope; Viral Envelope Proteins; Flavivirus; Zika Virus; Zika Virus Infection; Peptides; Dengue; Antiviral Agents; Phospholipids
PubMed: 37437754
DOI: 10.1016/j.bbamem.2023.184198 -
Nano Letters Jul 2017How complex cytoplasmic membrane proteins insert and fold into cellular membranes is not fully understood. One problem is the lack of suitable approaches that allow...
How complex cytoplasmic membrane proteins insert and fold into cellular membranes is not fully understood. One problem is the lack of suitable approaches that allow investigating the process by which polypeptides insert and fold into membranes. Here, we introduce a method to mechanically unfold and extract a single polytopic α-helical membrane protein, the lactose permease (LacY), from a phospholipid membrane, transport the fully unfolded polypeptide to another membrane and insert and refold the polypeptide into the native structure. Insertion and refolding of LacY is facilitated by the transmembrane chaperone/insertase YidC in the absence of the SecYEG translocon. Insertion into the membrane occurs in a stepwise, stochastic manner employing multiple coexisting pathways to complete the folding process. We anticipate that our approach will provide new means of studying the insertion and folding of membrane proteins and to mechanically reconstitute membrane proteins at high spatial precision and stoichiometric control, thus allowing the functional programming of synthetic and biological membranes.
Topics: Cell Membrane; Escherichia coli Proteins; Membrane Transport Proteins; Membranes, Artificial; Models, Molecular; Monosaccharide Transport Proteins; Phospholipids; Protein Binding; Protein Conformation; Protein Folding; Protein Transport; Stress, Mechanical; Symporters
PubMed: 28627175
DOI: 10.1021/acs.nanolett.7b01844 -
The Journal of Biological Chemistry May 2023N-retinylidene-phosphatidylethanolamine (N-Ret-PE), the Schiff-base conjugate formed through the reversible reaction of retinal (Vitamin A-aldehyde) and...
N-retinylidene-phosphatidylethanolamine (N-Ret-PE), the Schiff-base conjugate formed through the reversible reaction of retinal (Vitamin A-aldehyde) and phosphatidylethanolamine, plays a crucial role in the visual cycle and visual pigment photoregeneration. However, N-Ret-PE can react with another molecule of retinal to form toxic di-retinoids if not removed from photoreceptors through its transport across photoreceptor membranes by the ATP-binding-cassette transporter ABCA4. Loss-of-function mutations in ABCA4 are known to cause Stargardt disease (STGD1), an inherited retinal degenerative disease associated with the accumulation of fluorescent di-retinoids and severe loss in vision. A larger assessment of retinal-phospholipid Schiff-base conjugates in photoreceptors is needed, along with further investigation of ABCA4 residues important for N-Ret-PE binding. In this study we show that N-Ret-PE formation is dependent on pH and phospholipid content. When retinal is added to liposomes or photoreceptor membranes, 40 to 60% is converted to N-Ret-PE at physiological pH. Phosphatidylserine and taurine also react with retinal to form N-retinylidene-phosphatidylserine and N-retinylidene-taurine, respectively, but at significantly lower levels. N-retinylidene-phosphatidylserine is not a substrate for ABCA4 and reacts poorly with retinal to form di-retinoids. Additionally, amino acid residues within the binding pocket of ABCA4 that contribute to its interaction with N-Ret-PE were identified and characterized using site-directed mutagenesis together with functional and binding assays. Substitution of arginine residues and hydrophobic residues with alanine or residues implicated in STGD1 significantly reduced or eliminated substrate-activated ATPase activity and substrate binding. Collectively, this study provides important insight into conditions which affect retinal-phospholipid Schiff-base formation and mechanisms underlying the pathogenesis of STGD1.
Topics: Humans; ATP-Binding Cassette Transporters; Phosphatidylserines; Phospholipids; Retinoids; Stargardt Disease
PubMed: 36931393
DOI: 10.1016/j.jbc.2023.104614 -
Biochimica Et Biophysica Acta.... Oct 2020The plasma membrane phospholipid distribution of animal cells is markedly asymmetric. Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are concentrated in the... (Review)
Review
The plasma membrane phospholipid distribution of animal cells is markedly asymmetric. Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are concentrated in the inner leaflet, whereas phosphatidylcholine (PC) and sphingomyelin (SM) are concentrated in the outer leaflet. This non-equilibrium situation is maintained by lipid pumps (flippases or floppases), which utilize energy in the form of ATP to translocate lipids from one leaflet to the other. Scramblases, which are activated when physiologically required, transport lipids in both directions across the membrane and can abolish lipid asymmetry. Lipid asymmetry also causes imbalances in the areas occupied by lipid in the two membrane leaflets, contributing to membrane curvature. The asymmetry of PS across the plasma membrane plays a crucial signalling role in numerous physiological processes. Exposure of PS on the external surface of blood platelets stimulates blood coagulation. PS exposure by other cells during apoptosis provides an "eat me" signal to surrounding macrophages. Many peripheral and integral membrane proteins have polybasic PS-binding domains on their cytoplasmic surfaces which either provide a membrane anchor or affect activity. These domains can also determine trafficking within the cell and control regulation via an electrostatic switch mechanism, as well as potentially acting as "death sensors" when cytoplasmic PS is transferred to the extracellular leaflet during apoptosis. Apart from these physiological roles, external PS exposure by microorganisms, viruses and cancer cells allows them to mimic the immunosuppressive anti-inflammatory action of apoptotic cells and proliferate, thus providing a strong medical motivation for future research in the field of lipid asymmetry in membranes.
Topics: Animals; Cell Membrane; Humans; Phospholipids; Signal Transduction
PubMed: 32511979
DOI: 10.1016/j.bbamem.2020.183382 -
The Journal of Physical Chemistry... Dec 2022We calculated the free energies for calcium, magnesium, and zinc ions binding to a zwitterionic phospholipid bilayer by using molecular dynamics simulations and the...
We calculated the free energies for calcium, magnesium, and zinc ions binding to a zwitterionic phospholipid bilayer by using molecular dynamics simulations and the enhanced umbrella sampling technique. By decomposing the free energy into entropic and enthalpic contributions, we found that Ca has the highest binding affinity and that the overall process is endothermic combined with a secondary exothermic process at higher ion concentrations. The relatively low dehydration free energy of Ca allows it to release coordinated water upon binding to the membrane. The dehydrated Ca further coordinates with lipids, resulting in a weaker influence on the water orientation and increased entropy. However, when sufficient Ca ions are adsorbed, the concentrated cation layer induces a positive electrostatic field, which enhances the energy barrier for further ion binding and orients the adjacent water, resulting in decreased entropy. In contrast, binding of Mg and Zn is exothermic and less favored because they remain fully hydrated when interacting with lipids.
Topics: Phospholipids; Water
PubMed: 36448843
DOI: 10.1021/acs.jpclett.2c03019 -
Journal of Integrative Plant Biology Sep 2018Phospholipids, including phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS) and... (Review)
Review
Phospholipids, including phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS) and phosphoinositides, have emerged as an important class of cellular messenger molecules in various cellular and physiological processes, of which PA attracts much attention of researchers. In addition to its effect on stimulating vesicle trafficking, many studies have demonstrated that PA plays a crucial role in various signaling pathways by binding target proteins and regulating their activity and subcellular localization. Here, we summarize the functional mechanisms and target proteins underlying PA-mediated regulation of cellular signaling, development, hormonal responses, and stress responses in plants.
Topics: Phosphatidic Acids; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylinositols; Phosphatidylserines; Plant Development
PubMed: 29660254
DOI: 10.1111/jipb.12655 -
Role of flippases, scramblases and transfer proteins in phosphatidylserine subcellular distribution.Traffic (Copenhagen, Denmark) Jan 2015It is well known that lipids are heterogeneously distributed throughout the cell. Most lipid species are synthesized in the endoplasmic reticulum (ER) and then... (Review)
Review
It is well known that lipids are heterogeneously distributed throughout the cell. Most lipid species are synthesized in the endoplasmic reticulum (ER) and then distributed to different cellular locations in order to create the distinct membrane compositions observed in eukaryotes. However, the mechanisms by which specific lipid species are trafficked to and maintained in specific areas of the cell are poorly understood and constitute an active area of research. Of particular interest is the distribution of phosphatidylserine (PS), an anionic lipid that is enriched in the cytosolic leaflet of the plasma membrane. PS transport occurs by both vesicular and non-vesicular routes, with members of the oxysterol-binding protein family (Osh6 and Osh7) recently implicated in the latter route. In addition, the flippase activity of P4-ATPases helps build PS membrane asymmetry by preferentially translocating PS to the cytosolic leaflet. This asymmetric PS distribution can be used as a signaling device by the regulated activation of scramblases, which rapidly expose PS on the extracellular leaflet and play important roles in blood clotting and apoptosis. This review will discuss recent advances made in the study of phospholipid flippases, scramblases and PS-specific lipid transfer proteins, as well as how these proteins contribute to subcellular PS distribution.
Topics: Adenosine Triphosphatases; Animals; Biological Transport; Cell Membrane; Endoplasmic Reticulum; Humans; Phosphatidylserines; Phospholipids
PubMed: 25284293
DOI: 10.1111/tra.12233 -
Biochemical Society Transactions Jun 2020Cardiolipin (CL) and its precursor phosphatidylglycerol (PG) are important anionic phospholipids widely distributed throughout all domains of life. They have key roles... (Review)
Review
Cardiolipin (CL) and its precursor phosphatidylglycerol (PG) are important anionic phospholipids widely distributed throughout all domains of life. They have key roles in several cellular processes by shaping membranes and modulating the activity of the proteins inserted into those membranes. They are synthesized by two main pathways, the so-called eukaryotic pathway, exclusively found in mitochondria, and the prokaryotic pathway, present in most bacteria and archaea. In the prokaryotic pathway, the first and the third reactions are catalyzed by phosphatidylglycerol phosphate synthase (Pgps) belonging to the transferase family and cardiolipin synthase (Cls) belonging to the hydrolase family, while in the eukaryotic pathway, those same reactions are catalyzed by unrelated homonymous enzymes: Pgps of the hydrolase family and Cls of the transferase family. Because of the enzymatic arrangement found in both pathways, it seems that the eukaryotic pathway evolved by convergence to the prokaryotic pathway. However, since mitochondria evolved from a bacterial endosymbiont, it would suggest that the eukaryotic pathway arose from the prokaryotic pathway. In this review, it is proposed that the eukaryote pathway evolved directly from a prokaryotic pathway by the neofunctionalization of the bacterial enzymes. Moreover, after the eukaryotic radiation, this pathway was reshaped by horizontal gene transfers or subsequent endosymbiotic processes.
Topics: Archaea; Bacteria; Binding Sites; Biosynthetic Pathways; Cardiolipins; Catalysis; Eukaryota; Evolution, Molecular; Gene Transfer, Horizontal; Hydrolases; Mitochondria; Models, Molecular; Phosphatidylglycerols; Phospholipids; Phosphoric Monoester Hydrolases; Phylogeny
PubMed: 32490527
DOI: 10.1042/BST20190967 -
Acta Biochimica Polonica 2018Phosphatidic acid (PA) is the simplest glycerophospholipid naturally occurring in living organisms, and even though its content among other cellular lipids is minor, it... (Review)
Review
Phosphatidic acid (PA) is the simplest glycerophospholipid naturally occurring in living organisms, and even though its content among other cellular lipids is minor, it is drawing more and more attention due to its multiple biological functions. PA is a precursor for other phospholipids, acts as a lipid second messenger and, due to its structural properties, is also a modulator of membrane shape. Although much is known about interaction of PA with its effectors, the molecular mechanisms remain unresolved to a large degree. Throughout many of the well-characterized PA cellular sensors, no conserved binding domain can be recognized. Moreover, not much is known about the cellular dynamics of PA and how it is distributed among subcellular compartments. Remarkably, PA can play distinct roles within each of these compartments. For example, in the nucleus it behaves as a mitogen, influencing gene expression regulation, and in the Golgi membrane it plays a role in membrane trafficking. Here, we discuss how a biophysical experimental approach enabled PA behavior to be described in the context of a lipid bilayer and to what extent various physicochemical conditions may modulate the functional properties of this lipid. Understanding these aspects would help to unravel specific mechanisms of PA-driven membrane transformations and protein recruitment and thus would lead to a clearer picture of the biological role of PA.
Topics: Cell Compartmentation; Cell Membrane; Lipid Bilayers; Phosphatidic Acids
PubMed: 29913482
DOI: 10.18388/abp.2018_2592