Anionic Phosphatidic Acid Research Articles

The cationic nature of lysine-rich segments modulates the structural and biochemical properties of wild potato FSK3 dehydrin

Dehydrins are hydrophilic stress-induced proteins that are thought to protect cellular machinery from the adverse effect of dehydration caused by low temperature, drought, or salinity. In the previous study, acidic FSK3 dehydrin DHN24 from Solanum sogarandinum was found to accumulate at multiple sites in phloem cells in response to cold treatment. This study investigated the biochemical and structural properties of recombinant DHN24. It was shown that the overexpression of DHN24 in Escherichia coli led to the inhibition of bacterial growth. The purified DHN24 was found to protect lactate dehydrogenase from freeze-induced denaturation. Circular dichroism (CD) analysis showed that DHN24 was disordered in aqueous solutions, but adopted α-helical conformation in a membrane-mimetic environment using sodium dodecyl sulfate micelles. DHN24 also interacted with anionic phosphatidic acid (PA). DHN24 contains four lysine-rich regions including three K-segments and a region upstream of the S-segment. The role of their local cationic nature is unknown. These segments are predicted to form helical structures. The CD analysis of mutant proteins in the membrane-mimetic environment matched these predictions most closely, revealing that the positively charged lysine residues in these regions promoted disorder-to-order transitions. Moreover, the inhibition of bacterial growth and interactions with PA were regulated by the local cationic nature of DHN24, while no such regulation was observed for its cryoprotective activity. The importance of the positive charge of the lysine-rich segments and disordered structure for DHN24 activity is discussed in relation to its possible biological function.

How Tim proteins differentially exploit membrane features to attain robust target sensitivity

Immune surveillance cells such as T cells and phagocytes utilize integral plasma membrane receptors to recognize surface signatures on triggered and activated cells such as those in apoptosis. One such family of plasma membrane sensors, the transmembrane immunoglobulin and mucin domain (Tim) proteins, specifically recognize phosphatidylserine (PS) but elicit distinct immunological responses. The molecular basis for the recognition of lipid signals on target cell surfaces is not well understood. Previous results suggest that basic side chains present at the membrane interface on the Tim proteins might facilitate association with additional anionic lipids including but not necessarily limited to PS. We, therefore, performed a comparative quantitative analysis of the binding of the murine Tim1, Tim3, and Tim4, to synthetic anionic phospholipid membranes under physiologically relevant conditions. X-ray reflectivity and vesicle binding studies were used to compare the water-soluble domain of Tim3 with results previously obtained for Tim1 and Tim4. Although a calcium link was essential for all three proteins, the three homologs differed in how they balance the hydrophobic and electrostatic interactions driving membrane association. The proteins also varied in their sensing of phospholipid chain unsaturation and showed different degrees of cooperativity in their dependence on bilayer PS concentration. Surprisingly, trace amounts of anionic phosphatidic acid greatly strengthened the bilayer association of Tim3 and Tim4, but not Tim1. A novel mathematical model provided values for the binding parameters and illuminated the complex interplay among ligands. In conclusion, our results provide a quantitative description of the contrasting selectivity used by three Tim proteins in the recognition of phospholipids presented on target cell surfaces. This paradigm is generally applicable to the analysis of the binding of peripheral proteins to target membranes through the heterotropic cooperative interactions of multiple ligands.

Modulation of the potassium channel KcsA by anionic phospholipids: Role of arginines at the non-annular lipid binding sites

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites.Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines.

Special interaction of anionic phosphatidic acid promotes high secondary structure in tetrameric potassium channel.

Anionic phosphatidic acid (PA) has been shown to stabilize and bind stronger than phosphatidylglycerol via electrostatic and hydrogen bond interaction with the positively charged residues of potassium channel KcsA. However, the effects of these lipids on KcsA folding or secondary structure are not clear. In this study, the secondary structure analyses of KcsA potassium channel was carried out using circular dichroism spectroscopy. It was found that PA interaction leads to increases in α-helical and β-sheet content of KcsA protein. In PA, KcsA α-helical structure was further stabilized by classical membrane-active cosolvent trifluoroethanol followed by reduction in the β-sheet content indicating cooperative transformation from the β-sheet to an α-helical structure. The data further uncover the role of anionic PA in KcsA folding and provide mechanism by which strong hydrogen bonds/electrostatic interaction among PA headgroup and basic residues on lipid binding domains may induce high helical structure thereby altering the protein folding and increasing the stability of tetrameric assembly.

Complexation of cytochrome c with calixarenes incorporated into the lipid vesicles and supported membranes

We studied the interaction of cytochrome c (cyt c) with specific calixarenes (CX) incorporated into the large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) or supported lipid membranes (sBLM) and compared this with not specific adsorption of cyt c to the LUV containing DMPC and anionic phosphatidic acid (PA) or sBLM composed of a mixture of DMPC and dimyristoylphosphatidic acid (DMPA). We showed that with increasing concentration of CX the average size of LUV increased and zeta potential become more negative as it is suggested from dynamic light scattering experiments. For PA containing LUV the increase in vesicle diameter was less expressed, but zeta potential decreased similarly like that of LUV contained CX. Cyt c did not affect significantly the LUV size, but reduced the negative zeta potential both for CX and PA containing vesicles. Electrochemical impedance spectroscopy allowed us to determine binding of cyt c to sBLM contained CX or DMPA. In both cases we observed decrease of charge transfer resistance with increasing cyt c concentration. The analysis of binding process suggests that the main driving force for interaction of cyt c with sBLM is the negative surface charge.

Do Small Headgroups of Phosphatidylethanolamine and Phosphatidic Acid Lead to a Similar Folding Pattern of the K+ Channel?

Phospholipid headgroups act as major determinants in proper folding of oligomeric membrane proteins. The K+-channel KcsA is the most popular model protein among these complexes. The presence of zwitterionic nonbilayer lipid phosphatidylethanolamine (PE) is crucial for efficient tetramerization and stabilization of KcsA in a lipid bilayer. In this study, the influence of PE on KcsA folding properties was analyzed by tryptophan fluorescence and acrylamide quenching experiments and compared with the effect of anionic phosphatidic acid (PA). The preliminary studies suggest that the small size and hydrogen bonding capability of the PE headgroup influences KcsA folding via a mechanism quite similar to that observed for anionic PA.

Mechanism of mixed liposome solubilization in the presence of sodium dodecyl sulfate

Structural transformations of vesicles to micelles that take place during the interaction of sodium dodecyl sulfate (SDS) with individual and mixed vesicles of phosphatidyl choline (PC) and phosphatidic acid (PA), have been studied by monitoring changes in optical density and in the concentration of free SDS monomer in the respective systems. Incorporation of the surfactant monomers (SDS) in the bilayers was found to result in an initial increase in concentration of the mixed vesicles up to its saturation. Subsequently a progressive relaxation of these structures together with a simultaneous formation of mixed micelles was found to occur. The breakup of bilayer and the formation of mixed micelles were completely dependent on the structure of the individual phospholipid. The solubilization of the anionic phosphatidic acid vesicle was very fast in the presence of SDS, due to its simple structure and its compatibility with the SDS molecule. But solubilization of the zwitter ionic phosphatidyl choline vesicle was very complicated in the presence of SDS, possibly due to its complex structure and the zwitterionic nature. On the other hand, solubilization of the mixed vesicle (mixed liposome) and formation of mixed micelle was found to be relatively easier. This may be attributed to one of the phospholipid component preferentially coming out first through interaction with SDS, thereby making the overall system unstable and enhancing the micellization processes.

Calcium-Independent Activation of Protein Kinase C by the Dianionic Form of Phosphatidic Acid

Phosphatidic acid in the form of small unilamellar vesicles has a dissociation constant of about 8.3 as determined by 31P nuclear magnetic resonance (NMR) spectroscopy. The activation of protein kinase C (PKC) by monovalent phosphatidic acid or phosphatidylserine occurs only in the presence of Ca2+. However, PKC activity on membranes of divalent anionic phosphatidic acid is independent of Ca2+ concentration.

The role of calcium at the sarcolemma in the control of myocardial contractility.

The importance of sarcolemmal-bound calcium (Ca) in the control of contraction in mammalian myocardium is indicated by the following results. The curve that relates [Ca]o (from 50 microM to 10 mM) to force development and that which relates [Ca]o to Ca bound to a highly purified sarcolemmal fraction are superimposable. The ability of a series of cations to uncouple excitation from contraction is the same as their relative ability to displace Ca from the sarcolemma. Dimethonium, which specifically displaces cation from the diffuse double layer of the cellular surface, has little effect on contractile force. This indicates that the Ca actually bound to the sarcolemma is the surface Ca important in contractile control. Polymyxin B, a highly charged cationic amphiphilic peptidolipid, specifically competes for Ca-binding sites on anionic and zwitterionic phospholipid. It is a potent displacer of Ca from myocardial cells and purified sarcolemma and a potent uncoupler. Phospholipase D cleaves the nitrogenous base from sarcolemmal phospholipid with production of anionic phosphatidic acid. Phospholipase D treatment increases Ca bound to cells and purified sarcolemma and increases force development of ventricular tissue from both neonatal rat and adult rabbit. Insertion of charged amphiphiles in the sarcolemma as phospholipid analogues modulate interaction of Ca with the sarcolemma, e.g., anionic dodecylsulfate increases Ca bound to sarcolemmal vesicles by more than 80% and increases force development in rabbit papillary muscle by 100%. The effect of pH variation on Ca binding to phospholipid extracted from sarcolemma indicates that phospholipid accounts for at least 75% of the binding. The current model proposes a two-site control of Ca binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of sarcolemmal-bound calcium in regulation of myocardial contractile force

A rapidly exchangeable component of cellular calcium plays a significant role in the control of force development in heart muscle. Recently completed studies indicate that a large fraction of this calcium is bound to sites on the sarcolemma: The curve that relates extracellular calcium ([Ca]0)(from 50 microM to 10 mM) to force development and the one that relates [Ca]0 to calcium bound to a highly purified sarcolemmal fraction are superimposable. The ability of a series of cations (lanthanum, cadmium, manganese, magnesium) to uncouple excitation from contraction is the same as their relative ability to displace calcium from sarcolemma. Polymyxin B, a highly charged cationic amphiphilic peptidolipid, specifically competes for calcium-binding sites on anionic and zwitterionic phospholipid. It is a potent displacer of calcium from myocardial cells and purified sarcolemma and a potent uncoupler. Phospholipase D cleaves the nitrogenous base from sarcolemmal phospholipid with production of anionic phosphatidic acid. Phospholipase D treatment increases calcium bound to cells and to purified sarcolemma and increases force development of ventricular tissue from both neonatal rats and adult rabbits. Insertion of charged amphiphiles in the sarcolemma as phospholipid analogues modulates interaction of calcium with myocardial sarcolemma. Anionic dodecylsulfate increases calcium bound to sarcolemmal vesicles by more than 80% and increases force development in rabbit papillary muscle by 100%. Cationic dodecyltrimethylamine produces a negative effect on binding and decreases contractile force by 70%. Neutral laurylacetate produces negligible effects on binding and force development. The effect of pH variation on calcium binding to phospholipid extracted from sarcolemma indicates that the latter accounts for at least 75% of the binding.(ABSTRACT TRUNCATED AT 250 WORDS)