Synthesis of phospholipids containing photoactivatable carbene precursors in the headgroups and their crosslinking with membrane proteins

Many integral membrane enzymes require for their activity interactions with the polar headgroups of phospholipids, in addition to the hydrophobic interactions within the lipid bilayer. The interactions with the polar headgroups may have preferential or absolute specificity. To study such interactions, phospholipids have been synthesized which carry photoactivable moieties in their headgroups. Three types of phospholipids, PL-I, PL-II and PL-III, were synthesized. The synthetic phospholipids, PL-I and PL-II were able to reconstitute enzymatic activity of the membrane proteins which were studied. Covalent crosslinking between these phospholipids and the membrane proteins was demonstrated after photolysis of the reconstituted phospholipid-protein complexes.     

膜联蛋白: 植物生长过程中的多功能复合物

该文介绍了植物膜联蛋白(annexins)以及在生长过程中不同生理活动所扮演的角色,如钙离子通道的形成、膜融合、囊泡运输、信号转导和细胞骨架蛋白间的相互作用,以及可以结合F-肌动蛋白,具有过氧化物酶、离子通道,使ATP和GTP水解的功能。     

Lipid-protein interactions in biological membranes: a structural perspective

Lipid molecules bound to membrane proteins are resolved in some high-resolution structures of membrane proteins. An analysis of these structures provides a framework within which to analyse the nature of lipid-protein interactions within membranes. Membrane proteins are surrounded by a shell or annulus of lipid molecules, equivalent to the solvent layer surrounding a water-soluble protein. The lipid bilayer extends right up to the membrane protein, with a uniform thickness around the protein. The surface of a membrane protein contains many shallow grooves and protrusions to which the fatty acyl chains of the surrounding lipids conform to provide tight packing into the membrane. An individual lipid molecule will remain in the annular shell around a protein for only a short period of time. Binding to the annular shell shows relatively little structural specificity. As well as the annular lipid, there is evidence for other lipid molecules bound between the transmembrane α-helices of the protein; these lipids are referred to as non-annular lipids. The average thickness of the hydrophobic domain of a membrane protein is about 29 Å, with a few proteins having significantly smaller or greater thicknesses than the average. Hydrophobic mismatch between a membrane protein and the surrounding lipid bilayer generally leads to only small changes in membrane thickness. Possible adaptations in the protein to minimise mismatch include tilting of the helices and rotation of side chains at the ends of the helices. Packing of transmembrane α-helices is dependent on the chain length of the surrounding phospholipids. The function of membrane proteins is dependent on the thickness of the surrounding lipid bilayer, sometimes on the presence of specific, usually anionic, phospholipids, and sometimes on the phase of the phospholipid.     

Lipid-protein interactions in biological membranes: a structural perspective

Lipid molecules bound to membrane proteins are resolved in some high-resolution structures of membrane proteins. An analysis of these structures provides a framework within which to analyse the nature of lipid-protein interactions within membranes. Membrane proteins are surrounded by a shell or annulus of lipid molecules, equivalent to the solvent layer surrounding a water-soluble protein. The lipid bilayer extends right up to the membrane protein, with a uniform thickness around the protein. The surface of a membrane protein contains many shallow grooves and protrusions to which the fatty acyl chains of the surrounding lipids conform to provide tight packing into the membrane. An individual lipid molecule will remain in the annular shell around a protein for only a short period of time. Binding to the annular shell shows relatively little structural specificity. As well as the annular lipid, there is evidence for other lipid molecules bound between the transmembrane alpha-helices of the protein; these lipids are referred to as non-annular lipids. The average thickness of the hydrophobic domain of a membrane protein is about 29 A, with a few proteins having significantly smaller or greater thicknesses than the average. Hydrophobic mismatch between a membrane protein and the surrounding lipid bilayer generally leads to only small changes in membrane thickness. Possible adaptations in the protein to minimise mismatch include tilting of the helices and rotation of side chains at the ends of the helices. Packing of transmembrane alpha-helices is dependent on the chain length of the surrounding phospholipids. The function of membrane proteins is dependent on the thickness of the surrounding lipid bilayer, sometimes on the presence of specific, usually anionic, phospholipids, and sometimes on the phase of the phospholipid.     

Interaction of the K+ channel KcsA with membrane phospholipids as studied by ESI mass spectrometry

In this study we have used electrospray ionization mass spectrometry (ESI-MS) to investigate interactions between the bacterial K(+) channel KcsA and membrane phospholipids. KcsA was reconstituted into lipid vesicles of variable lipid composition. These vesicles were directly analyzed by ESI-MS or mixed with trifluoroethanol (TFE) before analysis. In the resulting mass spectra, non-covalent complexes of KcsA and phospholipids were observed with an interesting lipid specificity. The anionic phosphatidylglycerol (PG), and, to a lesser extent, the zwitterionic phosphatidylethanolamine (PE), which both are abundant bacterial lipids, were found to preferentially associate with KcsA as compared to the zwitterionic phosphatidylcholine (PC). These preferred interactions may reflect the differences in affinity of these phospholipids for KcsA in the membrane.     

Effects of lipids on mitochondrial functions

Mitochondria contain two membranes: the outer and inner membrane. Whereas the outer membrane is particularly enriched in phospholipids, the inner membrane has an unusual high protein content and forms large invaginations termed cristae. The proper phospholipid composition of the membranes is crucial for mitochondrial functions. Phospholipids affect activity, biogenesis and stability of protein complexes including protein translocases and respiratory chain supercomplexes. Negatively charged phospholipids such as cardiolipin are important for the architecture of the membranes and recruit soluble factors to the membranes to support mitochondrial dynamics. Thus, phospholipids not only form the hydrophobic core of biological membranes that surround mitochondria, but also create a specific environment to promote functions of various protein machineries. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

Membrane association and functional regulation of Sec3 by phospholipids and Cdc42

The exocyst is an octameric protein complex implicated in tethering post-Golgi secretory vesicles at the plasma membrane in preparation for fusion. However, it is not clear how the exocyst is targeted to and physically associates with specific domains of the plasma membrane and how its functions are regulated at those regions. We demonstrate that the N terminus of the exocyst component Sec3 directly interacts with phosphatidylinositol 4,5-bisphosphate. In addition, we have identified key residues in Sec3 that are critical for its binding to the guanosine triphosphate–bound form of Cdc42. Genetic analyses indicate that the dual interactions of Sec3 with phospholipids and Cdc42 control its function in yeast cells. Disrupting these interactions not only blocks exocytosis and affects exocyst polarization but also leads to defects in cell morphogenesis. We propose that the interactions of Sec3 with phospholipids and Cdc42 play important roles in exocytosis and polarized cell growth.     

脱脂菌紫质与天然紫膜水合变化的比较

本实验通过不同水合度下天然紫膜、脱脂菌紫质吸附等温线分析、红外光谱对比,讨论了天然紫膜小磷脂、蛋白质、水三者作用关系,认为磷脂对天然紫膜中蛋白质表而一些极性基团的分布及水合有重要作用,这些位点的水合对蛋白质进一步水合变化起重要作用.     

Anionic phospholipids are determinants of membrane protein topology.

The orientation of many membrane proteins is determined by the asymmetric distribution of positively charged amino acid residues in cytoplasmic and translocated loops. The positive-inside rule states that loops with large amounts of these residues tend to have cytoplasmic locations. Orientations of constructs derived from the inner membrane protein leader peptidase from Escherichia coli were found to depend on the anionic phospholipid content of the membrane. Lowering the contents of anionic phospholipids facilitated membrane passage of positively charged loops. On the other hand, elevated contents of acidic phospholipids in the membrane rendered translocation more sensitive to positively charged residues. The results demonstrate that anionic lipids are determinants of membrane protein topology and suggest that interactions between negatively charged phospholipids and positively charged amino acid residues contribute to the orientation of membrane proteins.     

Lipids trigger a conformational switch that regulates signal recognition particle (SRP)-mediated protein targeting

Co-translational protein targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for protein targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-targeting sequence. We show that the membrane-targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-protein interactions and their importance in the regulation of protein targeting and translocation across biological membranes.     

The Bilayer Collective Properties Govern the Interaction of an HIV-1 Antibody with the Viral Membrane

Efficient engagement with the envelope glycoprotein membrane-proximal external region (MPER) results in robust blocking of viral infection by a class of broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV). Developing an accommodation surface that engages with the viral lipid envelope appears to correlate with the neutralizing potency displayed by these bnAbs. The nature of the interactions established between the antibody and the lipid is nonetheless a matter of debate, with some authors arguing that anti-MPER specificity arises only under pathological conditions in autoantibodies endowed with stereospecific binding sites for phospholipids. However, bnAb-lipid interactions are often studied in systems that do not fully preserve the biophysical properties of lipid bilayers, and therefore, questions on binding specificity and the effect of collective membrane properties on the interaction are still open. Here, to evaluate the specificity of lipid interactions of an anti-MPER bnAb (4E10) in an intact membrane context, we determine quantitatively its association with lipid bilayers by means of scanning fluorescence correlation spectroscopy and all-atom molecular dynamic simulations. Our data support that 4E10 establishes electrostatic and hydrophobic interactions with the viral membrane surface and that the collective physical properties of the lipid bilayer influence 4E10 dynamics therein. We conclude that establishment of peripheral, nonspecific electrostatic interactions with the viral membrane through accommodation surfaces may assist high-affinity binding of HIV-1 MPER epitope at membrane interfaces. These findings highlight the importance of considering antibody-lipid interactions in the design of antibody-based anti-HIV strategies.     

General and specific lipid–protein interactions in Na,K-ATPase

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.”     

A modification of the split-tobacco etch virus method for monitoring interactions between membrane proteins in mammalian cells

Despite progress in the development of methods to monitor protein interactions, studies of interactions between membrane proteins in mammalian cells remain challenging. Protein complementation assays (PCAs) are commonly used to study interactions between proteins due to their simplicity. They are based on interaction-mediated reconstitution of a reporter protein, which can be easily monitored. Recently, a protein complementation method named split-TEV (tobacco etch virus) has been developed and is based on the functional reconstitution of TEV protease and subsequent proteolytic-mediated activation of reporters. In this work, we have developed a modification of the split-TEV method to study the interactions between membrane proteins with increased specificity. This assay was validated by addressing the interactions between different membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels. By comparing it with another PCA, we found that this new method showed a higher sensitivity.     

PLRP2 selectively localizes synaptic membrane proteins via acyl-chain remodeling of phospholipids

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.     

Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli

Gram-negative bacteria have an outer membrane (OM) that functions as a barrier to protect the cell from toxic compounds such as antibiotics and detergents. The OM is a highly asymmetric bilayer composed of phospholipids, glycolipids, and proteins. Assembly of this essential organelle occurs outside the cytoplasm in an environment that lacks obvious energy sources such as ATP, and the mechanisms involved are poorly understood. We describe the identification of a multiprotein complex required for the assembly of proteins in the OM of Escherichia coli. We also demonstrate genetic interactions between genes encoding components of this protein assembly complex and imp, which encodes a protein involved in the assembly of lipopolysaccharides (LPS) in the OM. These genetic interactions suggest a role for YfgL, one of the lipoprotein components of the protein assembly complex, in a homeostatic control mechanism that coordinates the overall OM assembly process.     

Calmodulin-dependent regulation of a lipid binding domain in the v-SNARE synaptobrevin and its role in vesicular fusion

Trans SNARE complex assembly is an essential step in Ca2+-dependent membrane fusion, although the SNARE proteins do not bind Ca2+ ions. Studies to evaluate how the Ca2+sensor protein calmodulin might regulate this process led to the identification of a consensus calmodulin binding motif in the v-SNARE VAMP2. This sequence (residues 77-90) is situated precisely C-terminal to the tetanus toxin (TeNT) and botulinum B toxin cleavage site (76Q-F77) close to the transmembrane anchor. The same domain also binds acidic phospholipids and Ca2+/calmodulin or lipid binding are mutually exclusive. Directed mutagenesis of basic or hydrophobic residues within this motif reduced interactions with both Ca2+/calmodulin and phospholipids to a similar extent. The effects of these mutations on Ca2+-dependent exocytosis was explored using an hGH release assay in permeabilized pheochromocytoma PC12 cells. Treatment of cells with tetanus toxin (TeNT), which cleaves endogenous VAMP, abolished secretion. Secretion could be re-established by transfecting TeNT-resistant VAMP with mutations (Q76V,F77W) in the cleavage site. However rescue of exocytosis was abolished when additional mutations (K83A,K87V or W89A,W90A) were introduced that inhibited calmodulin and phospholipid binding to VAMP. Thus calmodulin and/or phospholipid binding to the membrane proximal region of VAMP is required for Ca2+-dependent exocytosis. We speculate that interactions between cis phospholipids at the vesicle surface and the membrane proximal region of VAMP inhibits SNARE complex assembly. Displacement of these interactions by Ca2+/calmodulin may promote SNARE complex assembly and lead to trans interactions between the membrane proximal region of VAMP and phospholipids in the plasma membrane.     

Peroxisome assembly: matrix and membrane protein biogenesis

Mitochondria are dynamic organelles whose functional integrity requires a coordinated supply of proteins and phospholipids. Defined functions of specific phospholipids, like the mitochondrial signature lipid cardiolipin, are emerging in diverse processes, ranging from protein biogenesis and energy production to membrane fusion and apoptosis. The accumulation of phospholipids within mitochondria depends on interorganellar lipid transport between the endoplasmic reticulum (ER) and mitochondria as well as intramitochondrial lipid trafficking. The discovery of proteins that regulate mitochondrial membrane lipid composition and of a multiprotein complex tethering ER to mitochondrial membranes has unveiled novel mechanisms of mitochondrial membrane biogenesis.     

Protein myristoylation in protein-lipid and protein-protein interactions

Various proteins in signal transduction pathways are myristoylated. Although this modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown. Here we discuss the roles played by protein myristoylation, in both protein-lipid and protein-protein interactions. Myristoylation is involved in the membrane interactions of various proteins, such as MARCKS and endothelial NO synthase. The intermediate hydrophobic nature of the modification plays an important role in the reversible membrane anchoring of these proteins. The anchoring is strengthened by a basic amphiphilic domain that works as a switch for the reversible binding. Protein myristoylation is also involved in protein-protein interactions, which are regulated by the interplay between protein phosphorylation, calmodulin binding, and membrane phospholipids.     

Decoding P4-ATPase substrate interactions

Cellular membranes display a diversity of functions that are conferred by the unique composition and organization of their proteins and lipids. One important aspect of lipid organization is the asymmetric distribution of phospholipids (PLs) across the plasma membrane. The unequal distribution of key PLs between the cytofacial and exofacial leaflets of the bilayer creates physical surface tension that can be used to bend the membrane; and like Ca²⁺, a chemical gradient that can be used to transduce biochemical signals. PL flippases in the type IV P-type ATPase (P4-ATPase) family are the principle transporters used to set and repair this PL gradient and the asymmetric organization of these membranes are encoded by the substrate specificity of these enzymes. Thus, understanding the mechanisms of P4-ATPase substrate specificity will help reveal their role in membrane organization and cell biology. Further, decoding the structural determinants of substrate specificity provides investigators the opportunity to mutationally tune this specificity to explore the role of particular PL substrates in P4-ATPase cellular functions. This work reviews the role of P4-ATPases in membrane biology, presents our current understanding of P4-ATPase substrate specificity, and discusses how these fundamental aspects of P4-ATPase enzymology may be used to enhance our knowledge of cellular membrane biology.     

Proteomic analysis of blastema formation in regenerating axolotl limbs

Background

For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood.

Results

Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred.

Conclusion

This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents.