Role In Membrane Binding Research Articles

Effect of the number of sugar units on the interaction between diosgenyl saponin and membrane lipids

Saponin is the main bioactive component of the Dioscorea species, which are traditionally used for treating chronic diseases. An understanding of the interaction process of bioactive saponins with biomembranes provides insights into their development as therapeutic agents. The biological effects of saponins have been thought to be associated with membrane cholesterol (Chol). To shed light on the exact mechanisms of their interactions, we investigated the effects of diosgenyl saponins trillin (TRL) and dioscin (DSN) on the dynamic behavior of lipids and membrane properties in palmitoyloleolylphosphatidylcholine (POPC) bilayers using solid-state NMR and fluorescence spectroscopy. The membrane effects of diosgenin, a sapogenin of TRL and DSN, are similar to those of Chol, suggesting that diosgenin plays a major role in membrane binding and POPC chain ordering. The amphiphilicity of TRL and DSN enabled them to interact with POPC bilayers, regardless of Chol. In the presence of Chol, the sugar residues more prominently influenced the membrane-disrupting effects of saponins. The activity of DSN, which bears three sugar units, led to perturbation and further disruption of the membrane in the presence of Chol. However, TRL, which bears one sugar residue, increased the ordering of POPC chains while maintaining the integrity of the bilayer. This effect on the phospholipid bilayers is similar to that of cholesteryl glucoside. The influence of the number of sugars in saponin is discussed in more detail.

Topology of the SecA ATPase Bound to Large Unilamellar Vesicles

The soluble cytoplasmic ATPase motor protein SecA powers protein transport across the Escherichia coli inner membrane via the SecYEG translocon. Although dimeric in solution, SecA associates monomerically with SecYEG during secretion according to several crystallographic and cryo-EM structural studies. The steps SecA follows from its dimeric cytoplasmic state to its active SecYEG monomeric state are largely unknown. We have previously shown that dimeric SecA in solution dissociates into monomers upon electrostatic binding to negatively charged lipid vesicles formed from E. coli lipids. Here we address the question of the disposition of SecA on the membrane prior to binding to membrane embedded SecYEG. We mutated to cysteine, one at a time, 25 surface-exposed residues of a Cys-free SecA. To each of these we covalently linked the polarity-sensitive fluorophore NBD whose intensity and fluorescence wavelength-shift change upon vesicle binding report on the the local membrane polarity. We established from these measurements the disposition of SecA bound to the membrane in the absence of SecYEG. Our results confirmed that SecA is anchored in the membrane interface primarily by the positive charges of the N terminus domain. But we found that a region of the nucleotide binding domain II is also important for binding. Both domains are rich in positively charged residues, consistent with electrostatic interactions playing the major role in membrane binding. Selective replacement of positively charged residues in these domains with alanine resulted in weaker binding to the membrane, which allowed us to quantitate the relative importance of the domains in stabilizing SecA on membranes. Fluorescence quenchers inside the vesicles had little effect on NBD fluorescence, indicating that SecA does not penetrate significantly across the membrane. Overall, the topology of SecA on the membrane is consistent with the conformation of SecA observed in crystallographic and cryo-EM structures of SecA-SecYEG complexes, suggesting that SecA can switch between the membrane-associated and the translocon-associated states without significant changes in conformation.

Computational study of membrane binding and free energy of amphipathic helix (ALPS) on Osh4 to varied lipid bilayers

The Osh4 Amphipathic Lipid Packing Sensor (ALPS) peptide plays an important role in membrane binding to facilitate lipid transport. This work focuses on molecular simulations but is supplemented with wet lab experiments to probe the ALPS peptide binding to varied lipid bilayers for both the wildtype and selected mutations. The wet lab experiments are conducted in collaboration with Dr. Karlsson's Lab at U. Maryland. Our main findings are that the penetration depth of the peptide is enhanced by lower area per lipid membranes (DMPS and possibly DMPC vs.

Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the Drosophila Mitochondrial Replicative DNA Helicase.

Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from Drosophila melanogaster (Dm) has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the Dm mtDNA helicase. Further, we show that the NTD also serves a role in membrane binding. We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, Dm NUBPL, the known ISC donor for respiratory complex I and a putative donor for Dm mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.

Computational Prediction of Multi-Epitopes Vaccine from Envelope E Protein against Louping Ill Virus via Reverse Vaccinology

Louping ill disease is a zoonotic viral disease caused by louping ill virus in the genus Flavivirus. It belongs to the tick-borne flavivirus that is a part of the tick-borne encephalitis virus complex.The envelope E protein of louping ill virus is the major structural protein that plays an important role in membrane binding and inducing a protective immune response.The aim of the present study was to design multi epitopes vaccine from the envelope E glycoprotein against louping ill virus using immunoinformatic tools that elicited humoral and cellular immunity. Eighteen envelope E protein sequences were retrieved from NCBI and subjected to various immunoinformatics tools from IEDB to assess their conservancy, surface accessibility and antigenicity as promising epitopes against B cells. The binding affinity of the conserved predicted epitopes was analyzed against MHC-I and MHC-II alleles of the T cells. The predicted epitopes were further assessed for their population coverage. For B-cell 25, 18 and 12 epitopes were predicted as linear conserved epitopes, surface accessibility and antigenic respectively. However, nine epitopes overlapped all the B cell prediction tools. Among them three epitopes (205-TAEHLP-210,336-KPCR-339 and 349-SPDV-352) were proposed as B cell epitopes. For T cell, 75 epitopes were found to interact with MHC-I alleles. The epitopes 130-YVYDANKV-138and356-MLITPNPTI-364 were proposed as a peptide vaccine since they interacted with the highest number of MHC-1 alleles.Moreover a total of 195core epitopes were found to interact with MHC-II alleles. The core epitopes 130-YVYDANKV-138, 219-WFNDLALPW-227, 415-VIGEHAWDF-423 and 462-VALAWLGLN-470 interacted with higher number of MHC-II alleles and proposed as vaccine since they demonstrated high affinity to MHC-II alleles.The population coverage epitopes set for MHC-I and MHC-II alleles was 74.69% and 99.98%, respectively. While the epitopes set for all T cell, proposed epitopes was 100%. Nine epitopes were predicted eliciting B and T cells and proposed as vaccine candidates against louping ill virus. However, these proposed epitopes require clinical trials studies to ensure their efficacy as vaccine candidates.

A dynamic loop provides dual control over the catalytic and membrane binding activity of a bacterial serine hydrolase

The bacterial acyl protein thioesterase (APT) homologue FTT258 from the gram-negative pathogen Francisella tularensis exists in equilibrium between a closed and open state. Interconversion between these two states is dependent on structural rearrangement of a dynamic loop overlapping its active site. The dynamics and structural properties of this loop provide a simple model for how the catalytic activity of FTT258 could be spatiotemporally regulated within the cell. Herein, we characterized the dual roles of this dynamic loop in controlling its catalytic and membrane binding activity. Using a comprehensive library of loop variants, we determined the relative importance of each residue in the loop to these two biological functions. For the catalytic activity, a centrally located tryptophan residue (Trp66) was essential, with the resulting alanine variant showing complete ablation of enzyme activity. Detailed analysis of Trp66 showed that its hydrophobicity in combination with spatial arrangement defined its essential role in catalysis. Substitution of other loop residues congregated along the N-terminal side of the loop also significantly impacted catalytic activity, indicating a critical role for this loop in controlling catalytic activity. For membrane binding, the centrally located hydrophobic residues played a surprising minor role in membrane binding. Instead general electrostatic interactions regulated membrane binding with positively charged residues bracketing the dynamic loop controlling membrane binding. Overall for FTT258, this dynamic loop dually controlled its biological activities through distinct residues within the loop and this regulation provides a new model for the spatiotemporal control over FTT258 and potentially homologous APT function.

Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A.

Escherichia coli proline utilization A (EcPutA) is the archetype of trifunctional PutA flavoproteins, which function both as regulators of the proline utilization operon and bifunctional enzymes that catalyze the four-electron oxidation of proline to glutamate. EcPutA shifts from a self-regulating transcriptional repressor to a bifunctional enzyme in a process known as functional switching. The flavin redox state dictates the function of EcPutA. Upon proline oxidation, the flavin becomes reduced, triggering a conformational change that causes EcPutA to dissociate from the put regulon and bind to the cellular membrane. Major structure/function domains of EcPutA have been characterized, including the DNA-binding domain, proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase catalytic domains, and an aldehyde dehydrogenase superfamily fold domain. Still lacking is an understanding of the membrane-binding domain, which is essential for EcPutA catalytic turnover and functional switching. Here, we provide evidence for a conserved C-terminal motif (CCM) in EcPutA having a critical role in membrane binding. Deletion of the CCM or replacement of hydrophobic residues with negatively charged residues within the CCM impairs EcPutA functional and physical membrane association. Furthermore, cell-based transcription assays and limited proteolysis indicate that the CCM is essential for functional switching. Using fluorescence resonance energy transfer involving dansyl-labeled liposomes, residues in the α-domain are also implicated in membrane binding. Taken together, these experiments suggest that the CCM and α-domain converge to form a membrane-binding interface near the PRODH domain. The discovery of the membrane-binding region will assist efforts to define flavin redox signaling pathways responsible for EcPutA functional switching.

α-Synuclein and huntingtin exon 1 amyloid fibrils bind laterally to the cellular membrane.

Fibrillar aggregates involved in neurodegenerative diseases have the ability to spread from one cell to another in a prion-like manner. The underlying molecular mechanisms, in particular the binding mode of the fibrils to cell membranes, are poorly understood. In this work we decipher the modality by which aggregates bind to the cellular membrane, one of the obligatory steps of the propagation cycle. By characterizing the binding properties of aggregates made of α-synuclein or huntingtin exon 1 protein displaying similar composition and structure but different lengths to mammalian cells we demonstrate that in both cases aggregates bind laterally to the cellular membrane, with aggregates extremities displaying little or no role in membrane binding. Lateral binding to artificial liposomes was also observed by transmission electron microscopy. In addition we show that although α-synuclein and huntingtin exon 1 fibrils bind both laterally to the cellular membrane, their mechanisms of interaction differ. Our findings have important implications for the development of future therapeutic tools that aim to block protein aggregates propagation in the brain.

Specific Transient Interactions Between a Bacillus Virulence Factor and Phosphatidylcholine in Membranes

How do phospholipase Cs associated with bacterial virulence bind to membranes and search for substrates? The virulence factor Bacillus thuringiensis phosphatidylinositol‐specific phospholipase C (BtPI‐PLC) cleaves GPI‐anchored proteins off the outer leaflet of eukaryotic cells, helping the bacteria evade host defenses. Single molecule fluorescence microscopy studies of fluorescently labeled BtPI‐PLC interacting with model membranes reveal a koff of 2.64 ± 0.34 s‐1 at 22°C. This relatively fast dissociation is correlated with shallow anchorage of the protein in the membrane revealed by molecular dynamic simulations. Experimental and computational results suggest that cation‐π interactions between phosphatidylcholine (PC) headgroups and BtPI‐PLC Tyr residues play an important role in membrane binding. To further test this hypothesis, we substituted 3,5‐difluoroTyr for individual Tyr residues, thus perturbing cation‐π electrostatic interactions while increasing Tyr hydrophobicity. We also performed 31P NMR experiments with spin‐labeled BtPI‐PLC, to determine the location of PC binding sites on the protein surface. Both sets of experiments identify the same regions as important for PC binding. These biophysical and computational studies coupled with changes in enzyme activity provide a detailed, quantitative picture of how PC interactions with BtPI‐PLC influence its transient binding, PI cleavage and the search for substrates on the membrane.

In-vitro anti-proliferative and anti-oxidant activity of galangin, fisetin and quercetin: Role of localization and intermolecular interaction in model membrane

Flavonols are an important class of naturally occurring molecules and are known for their pharmacological activity. The activity is associated with the ability of flavonols to influence membrane-dependent processes. We have investigated the in-vitro anti-proliferative and anti-oxidant activity of galangin (GLN), fisetin (FTN) and quercetin (QTN), which possess variable number of phenolic hydroxyl groups. An attempt has been made to correlate the biological activity of these molecules with their interaction and localization in dipalmitoyl phosphatidyl choline (DPPC) bilayers, using differential dcanning calorimetry (DSC) and nuclear magnetic resonance (NMR) methods. Results indicate that GLN interacts to the alkyl chains of the lipid bilayer involving hydrophobic interactions. FTN and QTN interact with head region and sn-1-glycero region involving hydrogen bonding. Ring current induced chemical shifts of lipid protons, due to intermolecular interaction indicate that GLN acquires a parallel orientation with respect to the bilayer normal whereas FTN and QTN resume a mixed orientation. The membrane binding constants of these molecules are in the order GLN > QTN > FTN. It has been shown that the number and position of hydroxyl groups in these molecules play an important role in membrane binding and thereby in biological activity.

TBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation

Bid is a Bcl-2 family protein that promotes apoptosis by activating Bax and eliciting mitochondrial outer membrane permeabilization (MOMP). Full-length Bid is cleaved in response to apoptotic stimuli into two fragments, p7 and tBid (p15), that are held together by strong hydrophobic interactions until the complex binds to membranes. The detailed mechanism(s) of fragment separation including tBid binding to membranes and release of the p7 fragment to the cytoplasm remain unclear. Using liposomes or isolated mitochondria with fluorescently labeled proteins at physiological concentrations as in vitro models, we report that the two components of the complex quickly separate upon interaction with a membrane. Once tBid binds to the membrane, it undergoes slow structural rearrangements that result in an equilibrium between two major tBid conformations on the membrane. The conformational change of tBid is a prerequisite for interaction with Bax and is, therefore, a novel step that can be modulated to promote or inhibit MOMP. Using automated high-throughput image analysis in cells, we show that down-regulation of Mtch2 causes a significant delay between tBid and Bax relocalization in cells. We propose that by promoting insertion of tBid via a conformational change at the mitochondrial outer membrane, Mtch2 accelerates tBid-mediated Bax activation and MOMP. Thus the interaction of Mtch2 and tBid is a potential target for therapeutic control of Bid initiated cell death.

Structural location determines functional roles of the basic amino acids of KR-12, the smallest antimicrobial peptide from human cathelicidin LL-37

Cationic antimicrobial peptides are recognized templates for developing a new generation of antimicrobials to combat superbugs. Human cathelicidin LL-37 is an essential host defense molecule in human innate immunity. Previously, we identified KR-12 as the smallest antibacterial peptide of LL-37. KR-12 has a narrow activity spectrum since it is active against Gram-negative Escherichia coli but not Gram-positive Staphylococcus aureus. The functional roles of the basic amino acids of KR-12, however, have not yet been elucidated. An alanine scan of cationic amino acids of KR-12 provided evidence for their distinct roles in the activities of the peptides. Bacterial killing and membrane permeation experiments indicate that the R23A and K25A mutants, as well as the lysine-to-arginine mutant, were more potent than KR-12. Another three cationic residues (K18, R19, and R29) of KR-12, which are located in the hydrophilic face of the amphiphathic helix, appeared to be more important in clustering anionic lipids or hemolysis than R23 and K25 in the interfacial region. While the loss of interfacial R23 or K25 reduced peptide helicity, underscoring its important role in membrane binding, the overall increase in peptide activity of KR-12 could be ascribed to the increased peptide hydrophobicity that outweighed the role of basic charge in this case. In contrast, the mutations of interfacial R23 or K25 reduced peptide bactericidal activity of GF-17, an overlapping, more hydrophobic and potent peptide also derived from LL-37. Thus, the hydrophobic context of the peptide determines whether an alanine substitution of an interfacial basic residue increases or decreases membrane permeation and peptide activity.

Bordetella Adenylate Cyclase Toxin Can Bind Ganglioside GM1

Adenylate cyclase toxin (CyaA) is produced by Bordetella pertussis, the causative agent of whooping cough. CyaA specifically binds to the heavily Nglycosylated β2 integrin receptor CD11b/CD18 on its main cellular target myeloid phagocytic cells. However, CyaA can also interact with and intercalate into cell membranes lacking CD11b/CD18, where saccharides may play a propagating role in membrane binding. The present study was undertaken to examine whether CyaA can bind to ganglioside GM1 (GM1), a membrane raft associated oligosaccharidebearing lipid. For this sake, the potency of highly selective GM1 ligand cholera toxin subunit B (CTB) to block CyaA binding in GM1-positive CD11b/ CD18-negative human erythrocytes and K562 cells was studied by flow cytometry. Pre-treatment of erythrocytes and K562 cells with CTB markedly (29% and 28%, respectively) decreased their binding of CyaA, indicating that CyaA can bind to GM1, apparently by recognizing terminal galactose and sialic acid residues in GM1.

The role of electrostatic interactions in the membrane binding of melittin

The binding of melittin and the C-terminally truncated analogue of melittin (21Q) to a range of phospholipid bilayers was studied using surface plasmon resonance (SPR). The phospholipid model membranes included zwitterionic dimyristylphosphatidylcholine (DMPC) and dimyristylphosphatidylethanolamine (DMPE), together with mixtures DMPC/dimyristylphosphatidylglycerol (DMPG), DMPC/DMPG/cholesterol and DMPE/DMPG. Melittin bound rapidly to all membrane mixtures, whereas 21Q, which has a reduced charge, bound much more slowly on the DMPC and DMPC/DMPG mixtures reflecting the role of the initial electrostatic interaction. The loss of the cationic residues also significantly decreased the binding of 21Q with DMPC/DMPG/Cholesterol, DMPE and DMPE/DMPG. The role of electrostatics was also highlighted with NaCl in the buffer, which affected the way melittin bound to the different membranes, causing a more uniform, concentration dependant increase in response. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high α-helicity was associated with high binding affinity. Overall, the results demonstrate that the positively charged residues at the C-terminus of melittin play an essential role in membrane binding, that modulation of peptide charge influences selectivity of binding to different phospholipids and that manipulation of the cationic regions of antimicrobial peptides can be used to modulate membrane selectivity.

Interaction of Bacterial PI-Specific Phospholipase C With Lipid Bilayer Surfaces

PI-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is a 40 kDa enzyme that catalyzes the cleavage of GPI-anchored proteins and PI. PI-PLC binds to the membrane via the interfacial binding surface, a special region on the rim of the active site pocket. Hydrophobic and electrostatic interactions (via Trp and Lys residues, respectively) are proposed to play a role in membrane binding. To further examine these interactions, recombinant wild-type (WT) and mutant His6-tagged PI-PLCs (W47A, W242A, W47A/W242A and K44A) were expressed in E. coli and purified. Trp emission of WT and mutant enzymes did not undergo a blue shift upon addition of phosphatidylcholine (PC) liposomes, suggesting that PI-PLC does not deeply penetrate the membrane during membrane association. Trp quenching upon association of WT/mutant PI-PLC with bilayers containing brominated lipids (70% egg PC, 30% 6,7-Br2PC) was equal to that observed for pure egg PC, again suggesting no deep membrane penetration of the enzymes. Stopped-flow fluorescence studies showed first-order quenching of Trp fluorescence upon mixing of WT/mutant PI-PLCs with zwitterionic and negatively charged liposomes, consistent with the notion of a single binding event. The calculated dissociation constants revealed much weaker membrane association of W47A/W242A PI-PLC compared to WT enzyme, and ultimately suggest a role for these Trp residues in membrane association. Introduction of a PI substrate into egg PC liposomes resulted in 5-fold stronger association of WT and mutant PI-PLCs with the membrane surface. Lastly, Langmuir isotherms showed a change in the cross-sectional area per molecule of dimyristoyl-PC and dimyristoylphosphatidylglycerol monolayers upon injection of WT PI-PLC into the subphase, implying that PI-PLC association alters the surface packing of the monolayer. Changes in the area per molecule on PI-PLC binding were more substantial above the Tm of these lipids. (Supported by NSERC).

Study of hydrophobic interaction based binding of immunoglobulin G on synthetic membranes

Hydrophobic interaction based binding of immunoglobulin G (IgG) on synthetic membranes is relevant to areas such as bioseparations, immunoassays and biomaterials. Previous studies which focused on IgG binding on very hydrophobic surfaces are not unanimous on the orientation of the bound IgG molecule. In this paper we examine the mechanism of IgG binding on environment-responsive membranes possessing tunable hydrophobicity. Binding experiments were carried out in the membrane chromatography mode to avoid limitations associated with diffusional mass transport. Membrane-bound IgG was found to be capable of simultaneously binding both its antigen and Protein-A showing that the C-terminal region of Fc and the N-terminal region of Fab did not play any major role in membrane binding. This was confirmed by confocal laser scanning microscopy using fluorescent labeled proteins. The IgG molecule presumably interacted with the membrane surface primarily through its middle region consisting of the hinge and portion of upper Fc.

Membrane interactions of peptides representing the polybasic regions of three Rho GTPases are sensitive to the distribution of arginine and lysine residues

Rho GTPases are a multifunctional family of proteins that are localized at cellular membranes via an isoprenyl group covalently linked to a C-terminal cysteine. Close to this primary site of membrane anchoring there is often found an additional polybasic region (PBR), which plays a secondary role in membrane binding and targeting of the complex. Here, peptides derived from the PBRs of the Rho family proteins Rac1 (K(183)KRKRK), TCL (K(198)KKKKR) and Cdc42 (P(182)KKSRR) were prepared with hexalysine (K(6)) and hexaarginine (R(6)) to study their interactions with multilamellar vesicles of phosphatidylglycerol (DOPG) and headgroup-deuterated dimyristoylphosphatidylcholine (DMPC-d(4)) using (2)H and (31)P NMR. The membranes retained their lamellar architecture after peptide binding, but the (2)H NMR line shapes for DMPC-d(4) indicated that the bound peptides altered the orientation of the choline headgroups, consistent with a change in membrane surface charge. Rac1 and TCL peptides appeared to affect the headgroup orientation similarly to K(6), although the perturbations were weaker and unlike those induced by the Cdc42 peptide and R(6). Magic-angle spinning (31)P NMR spectra of the membranes showed significant and selective broadening of the peak for DMPC after addition of the peptides, with R(6) and the Cdc42 peptide having the greatest effect. The selective broadening may be a consequence of the lipids separating into short-lived domains enriched in peptide-bound DOPG and peptide-free DMPC. These results illustrate that a complex relationship exists between the sequence of PBRs and their behaviour at membrane surfaces, which may have implications for the cellular functions and localization of Rho GTPases.

Cholesterol and its anionic derivatives inhibit 5‐lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5-LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5-LO product synthesis.

Structural Determinants for Plant Annexin−Membrane Interactions

The interactions of two plant annexins, annexin 24(Ca32) from Capsicum annuum and annexin Gh1 from Gossypium hirsutum, with phospholipid membranes have been characterized using liposome-based assays and adsorption to monolayers. These two plant annexins show a preference for phosphatidylserine-containing membranes and display a membrane binding behavior with a half-maximum calcium concentration in the sub-millimolar range. Surprisingly, the two plant annexins also display calcium-independent membrane binding at levels of 10-20% at neutral pH. This binding is regulated by three conserved surface-exposed residues on the convex side of the proteins that play a pivotal role in membrane binding. Due to quantitative differences in the membrane binding behavior of N-terminally His-tagged and wild-type annexin 24(Ca32), we conclude that the N-terminal domain of plant annexins plays an important role, reminiscent of the findings in their mammalian counterparts. Experiments elucidating plant annexin-mediated membrane aggregation and fusion, as well as the effect of these proteins on membrane surface hydrophobicity, agree with findings from the membrane binding experiments. Results from electron microscopy reveal elongated rodlike assemblies of plant annexins in the membrane-bound state. It is possible that these structures consist of protein molecules directly interacting with the membrane surface and molecules that are membrane-associated but not in direct contact with the phospholipids. The rodlike structures would also agree with the complex data from intrinsic protein fluorescence. The tubular lipid extensions suggest a role in the membrane cytoskeleton scaffolding or exocytotic processes. Overall, this study demonstrates the importance of subtle changes in an otherwise conserved annexin fold where these two plant annexins possess distinct modalities compared to mammalian and other nonplant annexins.

C-terminal lysines determine phospholipid interaction of sarcomeric mitochondrial creatine kinase.

High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three C-terminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three C-terminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.