Membrane Binding Mode Research Articles

The crystal structure of the phosphatidylinositol 4-kinase IIα.

Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4-kinase type IIα (PI4K IIα), in complex with ATP solved by X-ray crystallography at 2.8 Å resolution. The structure revealed a non-typical kinase fold that could be divided into N- and C-lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C-lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane.

Functional Dissection of Toxoplasma gondii Perforin-like Protein 1 Reveals a Dual Domain Mode of Membrane Binding for Cytolysis and Parasite Egress

The recently discovered role of a perforin-like protein (PLP1) for rapid host cell egress by the protozoan parasite Toxoplasma gondii expanded the functional diversity of pore-forming proteins. Whereas PLP1 was found to be necessary for rapid egress and pathogenesis, the sufficiency for and mechanism of membrane attack were yet unknown. Here we further dissected the PLP1 knock-out phenotype, the mechanism of PLP1 pore formation, and the role of each domain by genetic complementation. We found that PLP1 is sufficient for membrane disruption and has a conserved mechanism of pore formation through target membrane binding and oligomerization to form large, multimeric membrane-embedded complexes. The highly conserved, central MACPF domain and the β-sheet-rich C-terminal domain were required for activity. Loss of the unique N-terminal extension reduced lytic activity and led to a delay in rapid egress, but did not significantly decrease virulence, suggesting that small amounts of lytic activity are sufficient for pathogenesis. We found that both N- and C-terminal domains have membrane binding activity, with the C-terminal domain being critical for function. This dual mode of membrane association may promote PLP1 activity and parasite egress in the diverse cell types in which this parasite replicates.

Fusogenicity of Naja naja atra cardiotoxin-like basic protein on sphingomyelin vesicles containing oxidized phosphatidylcholine and cholesterol

This study investigated the effect of oxidized phosphatidylcholine (oxPC) and cholesterol (Chol) on Naja naja atra cardiotoxin-like basic protein (CLBP)-induced fusion and leakage in sphingomyelin (SM) vesicles. Compared with those on PC/SM/Chol vesicles, CLBP showed a lower activity to induce membrane permeability but a higher fusogenicity on oxPC/SM/Chol vesicles. A reduction in inner-leaflet fusion elucidated that CLBP fusogenicity was not in parallel to its membrane-leakage activity on oxPC/SM/Chol vesicles. The lipid domain formed by Chol and SM supported CLBP fusogenicity on oxPC/SM/Chol vesicles, while oxPC altered the interacted mode of CLBP with oxPC/SM/Chol vesicles as evidenced by Fourier transform infrared spectra analyses and colorimetric phospholipid/polydiacetylene membrane assay. Although CLBP showed similar binding affinity with PC/SM/Chol and oxPC/SM/Chol vesicles, the binding capability of CLBP with PC/SM/Chol and oxPC/SM/Chol vesicles was affected differently by NaCl. This emphasized that CLBP adopted different membrane interaction modes upon binding with PC/SM/Chol and oxPC/SM/Chol vesicles. CLBP induced fusion in vesicles containing oxPC bearing the aldehyde group, and aldehyde scavenger methoxyamine abrogated the CLBP ability to induce oxPC/SM/Chol fusion. Taken together, our data indicate that Chol and oxPC bearing aldehyde group alter the CLBP membrane-binding mode, leading to fusogenicity promotion while reducing the membrane-damaging activity of CLBP.

Protein Kinase Cθ C2 Domain Is a Phosphotyrosine Binding Module That Plays a Key Role in Its Activation

Protein kinase Cθ (PKCθ) is a novel PKC that plays a key role in T lymphocyte activation. To understand how PKCθ is regulated in T cells, we investigated the properties of its N-terminal C2 domain that functions as an autoinhibitory domain. Our measurements show that a Tyr(P)-containing peptide derived from CDCP1 binds the C2 domain of PKCθ with high affinity and activates the enzyme activity of the intact protein. The Tyr(P) peptide also binds the C2 domain of PKCδ tightly, but no enzyme activation was observed with PKCδ. Mutations of PKCθ-C2 residues involved in Tyr(P) binding abrogated the enzyme activation and association of PKCθ with Tyr-phosphorylated full-length CDCP1 and severely inhibited the T cell receptor/CD28-mediated activation of a PKCθ-dependent reporter gene in T cells. Collectively, these studies establish the C2 domain of PKCθ as a Tyr(P)-binding domain and suggest that the domain may play a major role in PKCθ activation via its Tyr(P) binding.

Recognition Specificity of Proteins and Biomembranes: A Computational View

Cell membranes, including their individual components like membrane-bound proteins and particular lipids, attract a growing attention as very perspective pharmacological targets. Rational design of new efficient and selective compounds modulating activity of biomembranes, requires atomic-scale information on their spatial structure and dynamics under different conditions. Because such details resist easy experimental characterization, important insight can be gained via computer simulations.We present the results of structural/dynamic computational studies of membrane proteins and peptides with diverse fold, mode of membrane binding, and biological activities: antimicrobial and cell-penetrating peptides, cardiotoxins from snake venom, transmembrane domains of receptor tyrosine kinases. The computational approach combines Monte Carlo simulations in implicit membranes, molecular dynamics in full-atom lipid bilayers, and molecular hydrophobicity potential analysis. The predictive power of the method was proven via testing against high-resolution experimental data.Despite different structure and mechanism of membrane permeation, in all cases the polypeptide-membrane recognition reveals a prominent “self-adapting” character. Namely, the membrane active agents employ a wide arsenal of structural/dynamic tools in order to insert into lipid bilayer and to accomplish their function. Importantly, lipid bilayer of biological membranes plays essential role in the recognition/binding events. In particular, the membrane surface reveals highly dynamic lateral heterogeneities (clusters), which differ in their packing and hydrophobic properties from the bulk lipids. Such a mosaic nature of membranes is tuned in a wide range by the chemical nature and relative content of lipids, presence of ions, etc. This makes possible mutual adaptation of the two amphiphatic systems (peptide and membrane). Such a diversity of the factors important for polepeptide-bilayer interactions assures their efficient and robust binding to cell membranes. Understanding of such effects creates a basis for rational design of new physiologically active molecules and/or artificial membranes with predefined properties.

Sphingomyelin modulates interfacial binding of Taiwan cobra phospholipase A 2

The goal of the present study is to elucidate the effect of sphingomyelin on interfacial binding of Taiwan cobra phospholipase A 2 (PLA 2). Substitution of Asn-1 with Met caused a reduction in enzymatic activity and membrane-damaging activity of PLA 2 toward phospholipid vesicles, while sphingomyelin exerted an inhibitory effect on the biological activities of native and mutated PLA 2. Incorporation of sphingomyelin reduced membrane fluidity of phospholipid vesicles as evidenced by Laurdan fluorescence measurement. The results of self-quenching studies, binding of fluorescent probe, trinitrophenylation of Lys residues and fluorescence energy transfer between protein and lipid revealed that sphingomyelin altered differently membrane-bound mode of native and mutated PLA 2. Moreover, it was found that PLA 2 and N-terminally mutated PLA 2 adopted different conformation and geometrical arrangement on binding with membrane bilayer. Nevertheless, the binding affinity of PLA 2 and N-terminal mutant for phospholipid vesicles was not greatly affected by sphingomyelin. Together with the finding that mutation on N-terminus altered the gross conformation of PLA 2, our data indicate that sphingomyelin modulates the mode of membrane binding of PLA 2 at water/lipid interface, and suggest that the modulated effect of sphingomyelin depends on inherent structural elements of PLA 2.

Retroviral matrix and lipids, the intimate interaction

Retroviruses are enveloped viruses that assemble on the inner leaflet of cellular membranes. Improving biophysical techniques has recently unveiled many molecular aspects of the interaction between the retroviral structural protein Gag and the cellular membrane lipids. This interaction is driven by the N-terminal matrix domain of the protein, which probably undergoes important structural modifications during this process, and could induce membrane lipid distribution changes as well. This review aims at describing the molecular events occurring during MA-membrane interaction, and pointing out their consequences in terms of viral assembly. The striking conservation of the matrix membrane binding mode among retroviruses indicates that this particular step is most probably a relevant target for antiviral research.

The Conformation and Activity of Membane-Bound HIV Nef Studied by Neutron Reflectivity

Nef is one of six HIV-1 accessory proteins and directly contributes to AIDS progression. Nef has no catalytic activity but instead realizes its functions by interacting with cellular proteins. Nef is myristoylated on the N-terminus, associates with membranes, and may undergo a transition from a solution conformation to a membrane-associated conformation. It has been hypothesized that conformational rearrangement enables membrane-associated Nef to interact with cellular proteins. Despite its obvious disease importance, there is little or no direct information about the conformation of membrane-bound Nef. In this work we used neutron reflection to reveal details of the conformation of membrane-bound Nef. The conformation of membrane-bound Nef, and its ability to bind to the SH3 domain of Hck, was found to depend upon the mode of membrane binding. In solution Nef in known to bind the SH3 domain of Hck with high affinity (kd = 250 nM). In one mode, Nef was bound through an N-terminal His tag to Langmuir monolayers of DPPC mixed with a synthetic metal-chelating lipid (Biophys. J. 99, 1940, 2010). In that case, for a range of pH and salt concentration the core domain of membrane-bound Nef lies within a few angstroms of the lipid headgroups and a portion of the protein, presumably from the N-terminal arm, is inserted. In this conformation, Nef does not bind the SH3 domain of Hck. However, upon binding to negatively-charged membranes in absence of an interaction through the N-terminus, the core domain of Nef is displaced from the membrane, and binding of SH3 occurs. The ramifications of the these results for the actions of Nef in-vivo will be discussed.

Lipid Composition and Raft Domain Formation on the IAPP-Membrane Interaction: The Role of Cholesterol on the Inhibition of IAPP (Amylin) Fibrilization and the Reduction of Membrane Disruption in Model Liposomes and Mouse Pancreatic Islets

The misfolding and aggregation of islet amyloid polypeptide (IAPP) is known to exhibit an important role in the etiology of type-two diabetes mellitus. While it has been believed that the peptide becomes toxic in its large fibrous form, recent studies suggest that pre-fibril oligomers are in fact the predominantly toxic species. Permiabilization of IAPP on membranes is thus biphasic in nature. The latter phase is correlated with fiber formation and reaches maximal leakage. The degree of leakage, and hence membrane disruption, of the initial phase is dependent on the peptide to lipid ratio and percent anionic lipid in the membrane. Studies of IAPP toxicity have focused on POPC/POPS(G) vesicle systems. The homogeneity and anionic nature of the membranes have been shown to increase aggregation and toxicity. However, cellular membranes are vastly more complex. In this study, we investigate the in vitro interaction between IAPP and non-homogenous, cholesterol-containing model membranes; extend previous observations of the role of anionic phospholipids in membrane-catalyzed IAPP fibrilogenesis; and show that the presence of cholesterol dramatically inhibits IAPP fribrilogenesis and decreases membrane disruption in large unilamellar vesicles. Additionally, we demonstrate that human IAPP strongly permiablilizes raft type membranes. Fluorescence microscopy of islet cells shows increased IAPP toxicity after the removal of cholesterol from the membrane. These findings demonstrate that the mode of IAPP membrane binding and permiablization is highly dependent on the fluidity and phase of the membrane. The differences in this behavior may have significant implications in the development of type-two diabetes, as the change in membrane composition is dependent on a number of factors also related to the disease.

Large-Scale Computational Analysis of Protein Arrangement in the Lipid Bilayer

Spatial arrangements in the lipid bilayer of more than 1500 integral and peripheral membrane proteins with known 3D structures were calculated using a new continuum anisotopic solvent model of the lipid bilayer. The new model accounts for hydrophobic, hydrogen bonding, and long-range electrostatic solute-solvent interactions and includes the preferential solvation of protein groups by water. The model implements empirical dependencies of atomic solvation parameters and electrostatic energy terms on the bulk solvent properties including its dielectric constant (ε), hydrogen bonding acidity and basicity (α, β) and dipolarity/polarizability parameter (π∗). The dependencies were determined from 1269 partition coefficients of neutral molecules and ions from water to 19 solvents. The transmembrane profiles of polarity parameters (ε, α, β, π∗) in DOPC and DOPS bilayers were calculated based on published distributions of lipid fragments and water along the bilayer normal obtained in X-ray diffraction, neutron scattering and ESR studies. The obtained profiles indicate the existence of a “mid-polar” region (9 to 16 Å from the membrane center) that provides a significant energy gain from the hydrophobic interactions of nonpolar groups, but only a small electrostatic energy penalty associated with transfer of polar hydrogen-bonding groups and dipoles from water to this region. This explains the preferential accumulation of protein “hydrophobic dipoles” (aromatic rings of Trp and Tyr residues) in the “mid-polar” region. The results of the calculations have been included in the second version of the OPM database at opm.phar.umich.edu. The developed method and the database were applied for identification and functional assignment of potential membrane-associated proteins, planning mutagenesis experiments to verify the predicted membrane binding mode of peripheral proteins, prediction and interpretation of experimental membrane binding data.

Quantifying Protein Binding to Lipid Vesicles Using Fluorescence Correlation Spectroscopy (FCS)

Quantitative evaluation of protein binding to liposomes through the determination of their partition coefficient (Kp) is an important step in any lipid-protein interaction study because it allows the calculation of protein interfacial coverage of the lipid vesicles, often the critical parameter controlling the protein membrane binding mode (peripheral vs partial insertion) and/or its oligomerization state. In this study, the application of fluorescence correlation spectroscopy (FCS) technique to investigate the binding of protein molecules to phospholipid vesicles is presented. We have found that the type of fitting function used in the analysis of the experimental AC curves obtained in a partitioning experiment strongly influences the final recovered partition coefficients. We show that if the parameter chosen to establish the partition curves of the fluorescent peptide/ protein is the fractional amplitude associated with the slowest diffusing particles in the system (lipid vesicles) then it is necessary to consider explicitly the statistical (Poissonian) distribution of the fluorescently-labeled protein among the ensemble of lipid vesicles in the fitting procedure to not overestimate the determined Kp values. We have extended this analytical model by considering the presence of a trace amount of free fluorescent dye (nonbinding component) in the system to account for an apparent maximum binding level less than 100% in the experimental partitioning curves obtained for Alexa 488 fluorescently-labeled lysozyme and liposomes prepared with variable anionic phospholipid content. The extreme sensitivity of the FCS technique allowed uncoupling lysozyme partition from the protein-induced liposome aggregation, confirming that lysozyme binding to negatively charged liposomes is dominantly driven by electrostatic interactions.

Biophysical Characterization of Peptide Membrane Interactions and Membrane Permeabilization

Certain membrane-active amphipathic peptides, such as antimicrobial peptides, possess a characteristic feature of having several cationic amino acid residues at either N- or C-terminus. In an attempt to determine the functional significance of C-terminal cationic residues, we have studied the membrane binding mode and membrane pore formation by a 20-amino acid synthetic amphipathic peptide. Two cationic residues were replaced to Leu or Glu to see how the membrane binding and permeabilization activities of the peptide are modulated by charge neutralization and charge reversal mutations. The depth of membrane insertion of the wild type and mutant peptides was determined by measuring the quenching of the fluorescence of a single, C-terminally located tryptophan by dibromo-phosphatidylcholines brominated at various positions of the acyl chains, using pure POPC and 70% POPC + 30% POPG in lipid vesicles. In case of pure POPC membranes, Trp in all three peptides was located at around 9 Å from the membrane center, while for 30% POPG, Trp was inserted much deeper. Studies on peptide-mediated calcein release from phospholipid vesicles were conducted to see if the peptides are able to cause membrane pore formation and content release. Peptides with positively charged residues showed maximum calcein release for both lipid compositions whereas negative or hydrophobic residues were less effective in vesicle permeabilization. Preliminary ATR-FTIR experiments using both POPC and POPG have been performed to identify the orientation of the peptides within the lipid bilayer and their effect of the lipid order.

Mechanisms of Interfacial Activation of Human and Bee Venom Phospholipase A2 Enzymes

A class enzymes, known as interfacial enzymes, must bind to cell membranes in order to reach their full activity. Different mechanisms may underlie interfacial activation of functionally similar yet structurally divergent proteins. Human group IIA phospholipase A2 (hIIAPLA2), an inducible acute phase inflammatory enzyme found in blood and the synovial fluid, and group III bee venom PLA2 (bvPLA2) both catalyze the hydrolysis of the sn-2 acyl chain from membrane glycerophospholipids. Despite the functional similarity, they have been shown to employ distinct membrane binding modes and regulatory mechanisms (Pande et al., Biochemistry 45, 12436-12447, 2006). The aim of this work was to provide further insight into the molecular mechanisms of activation of hIIAPLA2 and bvPLA2, namely, to elucidate how phospholipid micelle formation affects their activities and identify conformational changes underlying activation. Activities of both enzymes were measured as a function of the concentration of a short-chain lipid, diheptanoylthio-phosphatidylcholine (DHTPC). The activity of bvPLA2 increased steadily with increasing DHTPC concentration, whereas the activity of hIIAPLA2 was negligible below the critical micelle concentration (cmc) of DHTPC and sharply increased above cmc. These data indicate that hIIAPLA2 behaves like a classical interfacial enzyme that undergoes activation upon micelle surface binding, and bvPLA2 is less sensitive to the aggregation state of the substrate. Circular dichroism studies indicated significant conformational changes in hIIAPLA2 upon activation by phospholipid micelle formation. Further studies will identify a correlation between conformational changes in both PLA2s and their activation and will thus enhance or understanding of the structural basis of interfacial activation.

Solution Structure and Membrane Binding of the Toxin Fst of theparAddiction Module

The par toxin−antitoxin system is required for the stable inheritance of the plasmid pAD1 in its native host Enterococcus faecalis. It codes for the toxin Fst and a small antisense RNA which inhibits translation of toxin mRNA, and it is the only known antisense regulated toxin−antitoxin system in Gram-positive bacteria. This study presents the structure of the par toxin Fst, the first atomic resolution structure of a component of an antisense regulated toxin−antitoxin system. The mode of membrane binding was determined by relaxation enhancements in a paramagnetic environment and molecular dynamics simulation. Fst forms a membrane-binding α-helix in the N-terminal part and contains an intrinsically disordered region near the C-terminus. It binds in a transmembrane orientation with the C-terminus likely pointing toward the cytosol. Membrane-bound, α-helical peptides are frequently found in higher organisms as components of the innate immune system. Despite similarities to these antimicrobial peptides, Fst shows neither hemolytic nor antimicrobial activity when applied externally to a series of bacteria, fungal cells, and erythrocytes. Moreover, its charge distribution, orientation in the membrane, and structure distinguish it from antimicrobial peptides.

New Insights into the Interfacial Activation of Secreted Phospholipase A2

Despite numerous studies towards elucidation of the structural basis of activation of secreted PLA2s upon membrane binding (interfacial activation), no consistent or clear picture has emerged thus far. Previously we have reported significant changes in the secondary and dynamic structures of human group IB and IIA PLA2s, as well as changes in their mode of membrane binding during activation. Here we have conducted atomic resolution NMR studies on free and phospholipid micelle-bound human group IIA PLA2 (hIIAPLA2) to detect more detailed molecular events underlying interfacial activation. Two-dimensional 1H,15N-HSQC spectra have been obtained at 600 MHz on Ca2+-free and Ca2+-loaded hIIAPLA2 in the presence of dodecylphosphocholine (DPC) micelles. Upon complex formation with the micelles, signals from arginine side chain -NH2 groups of Ca2+-loaded hIIAPLA2 are observed, whereas for Ca2+-free PLA2 these signals are absent because of fast H/D exchange with the solvent. This suggests that the Ca2+-loaded hIIAPLA2 tightly binds to the micelles so these groups are sequestered at the PLA2-micellar interface and shielded from the solvent, or that they are otherwise stabilized by strong hydrogen bonding in the micelle-bound state. TROSY experiments (900 MHz) on Ca2+-loaded, 15N,13C-labeled hIIAPLA2 in the absence and presence of DPC micelles (1:600 protein-to-DPC molar ratio) identify substantial conformational changes in PLA2 upon binding to the micelles. Based on the assigned chemical shifts, important structural changes occur throughout the protein. The molecular mechanism of the strong increase in activity of hIIAPLA2 upon phospholipid surface binding is likely to involve a widening of the substrate binding pocket, mediated by a rigid-body movement of the N-terminal helix via interactions of the cationic residues (e.g., Arg7) with lipid phosphate groups. This mechanism will be tested in further studies and may be shared by other secreted PLA2 isoforms.

Membrane binding mode of intrinsically disordered cytoplasmic domains of T cell receptor signaling subunits depends on lipid composition

Intrinsically disordered cytoplasmic domains of T cell receptor (TCR) signaling subunits including ζ cyt and CD3ε cyt all contain one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor triggering. Membrane binding-induced helical folding of ζ cyt and CD3ε cyt ITAMs is thought to control TCR activation. However, the question whether or not lipid binding of ζ cyt and CD3ε cyt is necessarily accompanied by a folding transition of ITAMs remains open. In this study, we investigate whether the membrane binding mechanisms of ζ cyt and CD3ε cyt depend on the membrane model used. Circular dichroic and fluorescence data indicate that binding of ζ cyt and CD3ε cyt to detergent micelles and unstable vesicles is accompanied by a disorder-to-order transition, whereas upon binding to stable vesicles these proteins remain unfolded. Using electron microscopy and dynamic light scattering, we show that upon protein binding, unstable vesicles fuse and rupture. In contrast, stable vesicles remain intact under these conditions. This suggests different membrane binding modes for ζ cyt and CD3ε cyt depending on the bilayer stability: (1) coupled binding and folding, and (2) binding without folding. These findings explain the long-standing puzzle in the literature and highlight the importance of the choice of an appropriate membrane model for protein–lipid interactions studies.

Despite a Conserved Cystine Knot Motif, Different Cyclotides Have Different Membrane Binding Modes

Cyclotides are cyclic proteins produced by plants for defense against pests. Because of their remarkable stability and diverse bioactivities, they have a range of potential therapeutic applications. The bioactivities of cyclotides are believed to be mediated through membrane interactions. To determine the structural basis for the biological activity of the two major subfamilies of cyclotides, we determined the conformation and orientation of kalata B2 (kB2), a Möbius cyclotide, and cycloviolacin O2 (cO2), a bracelet cyclotide, bound to dodecylphosphocholine micelles, using NMR spectroscopy in the presence and absence of 5- and 16-doxylstearate relaxation probes. Analysis of binding curves using the Langmuir isotherm indicated that cO2 and kB2 have association constants of 7.0 × 10 3 M −1 and 6.0 × 10 3 M −1, respectively, consistent with the notion that they are bound near the surface, rather than buried deeply within the micelle. This suggestion is supported by the selective broadening of micelle-bound cyclotide NMR signals upon addition of paramagnetic Mn ions. The cyclotides from the different subfamilies exhibited clearly different binding orientations at the micelle surface. Structural analysis of cO2 confirmed that the main element of the secondary structure is a β-hairpin centered in loop 5. A small helical turn is present in loop 3. Analysis of the surface profile of cO2 shows that a hydrophobic patch stretches over loops 2 and 3, in contrast to the hydrophobic patch of kB2, which predominantly involves loops 2 and 5. The different location of the hydrophobic patches in the two cyclotides explains their different binding orientations and provides an insight into the biological activities of cyclotides.

Disulfide bond as a structural determinant of prion protein membrane insertion.

Conversion of the normal soluble form of prion protein, PrP (PrP(C)), to proteinase K-resistant form (PrP(Sc)) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from alpha-helix to beta-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111-135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.

Effects of Lipid Phase Transition and Membrane Surface Charge on the Interfacial Activation of Phospholipase A2

Phospholipase A2 (PLA2) enzymes act at the membrane-water interface to access their phospholipid substrate from the membrane. They are regulated by diverse factors, including the membrane charge, fluidity, mode of membrane binding (insertion, orientation), and allosteric conformational effects. Relative contributions of these factors to the complex kinetics of PLA2 activation are not well understood. Here we examine the effects of thermal phase transitions and the surface charge of phospholipid membranes on the activation of human pancreatic PLA2. The temperature dependence of the initial catalytic rate of PLA2 peaks around the lipid phase transition temperature (Tm) when Tm is not too far from physiological temperatures (30-40 degrees C), and the peak is higher in the presence of anionic membranes. High PLA2 activity can be induced by thermal perturbations of the membrane. Temperature-dependent fluorescence quenching experiments show that despite dramatic effects of the lipid phase transition on PLA2 activity, the membrane insertion depth of PLA2 increases only modestly above Tm. The data show that membrane structural disorder, and not the depth of membrane insertion, plays a major role in PLA2 activity.

The Membrane-proximal Fusion Domain of HIV-1 GP41 Reveals Sequence-specific and Fine-tuning Mechanism of Membrane Binding

The membrane interface-partitioning region preceding the transmembrane anchor of the human immunodeficiency virus type 1 (HIV-1) gp41 envelope protein is one of the sites responsible for virus binding to its host cell membrane and subsequent fusion events. Here, we used molecular modeling techniques to assess membrane interactions, structure, and hydrophobic properties of the fusion-active peptide representing this region, several of its homologs from different HIV-1 strains, as well as a peptide—defective gp41 phenotype—unable to mediate cell-cell fusion and virus entry. It is shown that the wild-type peptides bind to the water-membrane interface in α-helical conformation, while the mutant adopts partly destabilized helix-break-helix structure on the membrane surface. The wild-type peptides reveal specific “tilted oblique-oriented” pattern of hydrophobicity on their surfaces—the property specific for fusion regions of other viruses. Fusion peptides penetrate into the membrane with their N-termini and reveal “fine-tuning” interactions with membrane and water environments: the shift of this balance (e.g., due to point mutations) may dramatically change the mode of membrane binding, and therefore, may cause loss of fusion activity. The modeling results agree well with experimental data and provide a strategy to delineate fusogenic regions in amino acid sequences of viral proteins.