No abstract

The local generation of phosphatidic acid plays a key role in the regulation of intracellular membrane transport through mechanisms which are largely unknown. Phosphatidic acid may recruit and activate downstream effectors, or change the biophysical properties of the membrane and directly induce membrane bending and/or destabilization. To evaluate these possibilities, we determined the phase properties of phosphatidic acid and lysophosphatidic acid at physiological conditions of pH and ion concentrations. In single-lipid systems, unsaturated phosphatidic acid behaved as a cylindrical, bilayer-preferring lipid at cytosolic conditions (37°C, pH 7.2, 0.5 mM free Mg 2+ ), but acquired a type-II shape at typical intra-Golgi conditions, a mildly acidic pH and submillimolar free Ca 2+ (pH 6.6±5.9, 0.3 mM Ca 2+ ). Lysophosphatidic acid formed type-I lipid micelles in the absence of divalent cations, but anhydrous cationlysophosphatidic acid bilayer complexes in their presence. These data suggest a similar molecular shape for phosphatidic acid and lysophosphatidic acid at cytosolic conditions; however, experiments in mixed-lipid systems indicate that their shape is not identical. Lysophosphatidic acid stabilized the bilayer phase of unsaturated phosphatidylethanolamine, while the opposite effect was observed in the presence of phosphatidic acid. These results support the hypothesis that a conversion of lysophosphatidic acid into phosphatidic acid by endophilin or BARS (50 kDa brefeldin A ribosylated substrate) may induce negative spontaneous monolayer curvature and regulate endocytic and Golgi membrane fission. Alternative models for the regulation of membrane fission based on the strong dependence of the molecular shape of (lyso)phosphatidic acid on pH and divalent cations are also discussed.

In this study the interaction between the glycoalkaloids alpha-chaconine, alpha-solanine and alpha-tomatine and sterols in model membranes was analysed systematically using techniques like membrane leakage, binding experiments, detergent extraction, electron microscopy, NMR and molecular modelling. The most important properties for sterols to interact with glycoalkaloids turned out to be a planer ring structure and a 3 beta-OH group, whereas for alpha-chaconine the 5-6 double bond and the 10-methyl group were also of importance. The importance of sugar-sugar interactions was illustrated by the high synergistic effect between alpha-chaconine and alpha-solanine, the leakage enhancing effect of glycolipids, and the almost complete loss of activity after deleting one or more mono-saccharides from the glycoalkaloids. The formed complexes which were resistant against detergent extraction existed of glycoalkaloid/sterol in a 1:1 ratio and formed tubular structures (alpha-chaconine) with an inner monolayer of phospholipids, whereas with alpha-tomatine also spherical structures were formed. Based on the results a molecular model for glycoalkaloid induced membrane disruption is presented.

The formation of phosphatidic acid (PA) from lysophosphatidic acid (LPA), diacylglycerol, or phosphatidylcholine plays a key role in the regulation of intracellular membrane fission events, but the underlying molecular mechanism has not been resolved. A likely possibility is that PA affects local membrane curvature facilitating membrane bending and fission. To examine this possibility, we determined the spontaneous radius of curvature (R 0p ) of PA and LPA, carrying oleoyl fatty acids, using well-established X-ray diffraction methods. We found that, under physiological conditions of pH and salt concentration (pH 7.0, 150 mM NaCl), the R 0p values of PA and LPA were -46 Å and +20 Å, respectively. Thus PA has considerable negative spontaneous curvature while LPA has the most positive spontaneous curvature of any membrane lipid measured to date. The further addition of Ca 2+ did not significantly affect lipid spontaneous curvature; however, omitting NaCl from the hydration buffer greatly reduced the spontaneous curvature of PA, turning it into a cylindrically shaped lipid molecule (R 0p of -1.3 × 10 2 Å). Our quantitative data on the spontaneous radius of curvature of PA and LPA at a physiological pH and salt concentration will be instrumental in developing future models of biomembrane fission.

Phosphatidic acid and lysophosphatidic acid are minor but important anionic bioactive lipids involved in a number of key cellular processes, yet these molecules have a simple phosphate headgroup.To find out what is so special about these lipids, we determined the ionization behavior of phosphatidic acid (PA) and lysophosphatidic acid (LPA) in extended (flat) mixed lipid bilayers using magic angle spinning 31 P NMR. Our data show two surprising results. First, despite identical phosphomonoester headgroups, LPA carries more negative charge than PA when present in a phosphatidylcholine bilayer. Dehydroxy-LPA [1-oleoyl-3-(phosphoryl)propanediol] behaves in a manner identical to that of PA, indicating that the difference in negative charge between LPA and PA is caused by the hydroxyl on the glycerol backbone of LPA and its interaction with the phosphomonoester headgroup. Second, deprotonation of phosphatidic acid and lysophosphatidic acid was found to be strongly stimulated by the inclusion of phosphatidylethanolamine in the bilayer, indicating that lipid headgroup charge depends on local lipid composition and will vary between the different subcellular locations of (L)PA. Our findings can be understood in terms of a hydrogen bond formed within the phosphomonoester headgroup of (L)PA and its destabilization by competing intra-or intermolecular hydrogen bonds. We propose that this hydrogen bonding property of (L)PA is involved in the various cellular functions of these lipids.Phosphatidic acid (PA) 1 and the related lipid lysophosphatidic acid (LPA) are important minor lipid species in the cell. They are involved in many intracellular processes, and are important intermediates in lipid biosynthesis (1). For example, binding of LPA to its receptors evokes various cellular responses, and the local formation of (L)PA is part of signaling cascades, in particular in the regulation of membrane dynamics such as fusion and fission events, either indirectly through the recruitment of downstream effectors or directly by mediating (local) changes in the biophysical properties of the membrane (2-12).PA and LPA have a relatively simple chemical structure consisting of only a glycerol, one (LPA) or two (PA) acyl chains, and a phosphate, and it is interesting to note that these simple phospholipids are involved in such diverse processes, and are able to bind specifically to so many different types of proteins (3, 13). The question then is what is so special about these lipids. An obvious suggestion relates to the phosphate headgroup, which is attached to the glycerol backbone as a phosphomonoester, a unique feature of these lipids. Phosphomonoesters have two pK a 's, one of which is expected to be in the physiological pH range. As a consequence, small changes in (physiological) pH will affect the charge and influence the molecular shape and lipid phase behavior of these lipids (14,15). Under physiological conditions at neutral pH, phosphatidic acid is a cone (type II)-shaped lipid with a negative spontaneous curvature clos...

Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed ␣-helical bundle through the N-terminal tandem GG4-like motif Thr 652 -X 3 -Ser 656 -X 3 -Gly 660 , providing an explanation for the pathogenic power of some oncogenic mutations.The epidermal growth factor receptor (or ErbB) family is an important class of receptor tyrosine kinases involved in transmission of biochemical signals governing cell fate (1). Four human ErbB family members form numerous homo-and heterodimer combinations and bind different epidermal growth factor-related ligands, thus performing diverse functions in a complex signaling network (2). The binding of peptide growth factors to the extracellular domain of the receptor triggers the dimerization of receptor monomers or a change in the relative orientation of monomers in preformed receptor dimers, leading to autophosphorylation of tyrosine residues in the cytoplasmic kinase domain (3, 4). Biochemical and genetic studies have revealed that the single-helix transmembrane (TM) 3 domains of ErbB play an active role in the dimerization process and associate strongly in the absence of extracellular ligand-binding and cytoplasmic kinase domains (5, 6). Mutational analysis assumed that the dimerization involves consensus small-X 3 -small (so-called GG4-like) motifs, formed by residues with small side chains allowing tight helix packing (7-9). Receptor tyrosine kinase TM sequences often contain several remote GG4-like motifs, suggesting the ability of their TM domains to adopt more than one conformation, e.g. upon so-called rotation-coupled activation of the receptor (4, 10, 11). Recent molecular modeling and solid-state NMR studies performed to predict the spatial structures of the dimeric TM domains of the human ErbB2 receptor and its rat homolog have disclosed two possible dimer conformations with interfaces located at either the N or C terminus of the ␣-helical TM segment, employing different GG4-like motifs for dimerization (7,(11)(12)(13). Nevertheless, an experimental spatial structure of the dimeric TM domain for ErbB2 as well as for any other receptor tyrosine kinase family members has not been reported so far.Here, we present the high resolution structure of the homodimeric ErbB2 TM domain in a membrane-mimicking lipid environment solved by a heteronuclear NMR technique combined with molecular dynamics (MD) relaxation in an explicit membrane. Our results distinguish one of the potential conformations of the homodimer, which can be ascribed to the active state of the tyrosine kinase. On the basis of the analysis of the local conformation of the dimerization interface, we propose a molecular mechanism of actio...

Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 fromKrokinobacter eikastuswas discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics.

The potassium channel KcsA forms an extremely stable tetramer. Despite this high stability, it has been shown that the membrane-mimicking solvent 2,2,2-trifluoroethanol (TFE) can induce tetramer dissociation [Valiyaveetil, F. I., et al. (2002) Biochemistry 41, 10771-7, and Demmers, J. A. A., et al. (2003) FEBS Lett. 541, 69-77]. Here we have studied the effect of TFE on the structure and oligomeric state of the KcsA tetramer, reconstituted in different lipid systems. It was found that TFE changes the secondary and tertiary structure of KcsA and that it can dissociate the KcsA tetramer in all systems used. The tetramer is stabilized by a lipid bilayer as compared to detergent micelles. The extent of stabilization was found to depend on the nature of the lipids: a strong stabilizing effect of the nonbilayer lipid phosphatidylethanolamine (PE) was observed, but no effect of the charged phoshosphatidylglycerol (PG) as compared to phosphatidylcholine (PC) was found. To understand how lipids stabilize KcsA against TFE-induced tetramer dissociation, we also studied the effect of TFE on the bilayer organization in the various lipid systems, using (31)P and (2)H NMR. The observed lipid dependency was similar as was found for tetramer stabilization: PE increased the bilayer stability as compared to PC, while PG behaved similar to PC. Furthermore, it was found that TFE has a large effect on the acyl chain ordering. The results indicate that TFE inserts primarily in the membrane interface. We suggest that the lipid bilayer stabilizes the KcsA tetramer by the lateral pressure in the acyl chain region and that this stabilizing effect increases when a nonbilayer lipid like PE is present.