Lipid-mimetic Environment Research Articles

Structural dynamics of Zika virus NS1 via a reductionist approach reveal the disordered nature of its β-roll domain in isolation

Flavivirus Non-structural 1 (NS1) protein performs multiple functions and it is highly plausible that significant structural and folding dynamics of NS1 might play a role in its multifunctionality. It is important to understand the structural conformations of NS1 and its domains in isolation, possibly highlighting the implications on the overall NS1 protein dynamics. Therefore, we have employed extensively long molecular dynamic (MD) simulations in understanding the dynamics of the three structural domains (i.e., β-roll, wing, and β-ladder) in isolation, as a reductionist approach. We also found that the β-ladder domain is highly flexible, while the β-roll domain is disordered during long simulations. Further, we experimentally validated our findings using CD spectroscopy and confirmed the intrinsically disordered behavior of NS1 β-roll in isolation and lipid mimetic environments. Therefore, we believe this study may have implications for significant dynamics played by NS1 protein, specifically during oligomerization of NS1.

Multipurpose peptides: The venoms of Amazonian stinging ants contain anthelmintic ponericins with diverse predatory and defensive activities

In the face of increasing drug resistance, the development of new anthelmintics is critical for controlling nematodes that parasitise livestock. Although hymenopteran venom toxins have attracted attention for applications in agriculture and medicine, few studies have explored their potential as anthelmintics. Here we assessed hymenopteran venoms as a possible source of new anthelmintic compounds by screening a panel of ten hymenopteran venoms against Haemonchus contortus, a major pathogenic nematode of ruminants. Using bioassay-guided fractionation coupled with liquid chromatography-tandem mass spectrometry, we identified four novel anthelmintic peptides (ponericins) from the venom of the neotropical ant Neoponera commutata and the previously described ponericin M−PONTX−Na1b from Neoponera apicalis venom. These peptides inhibit H. contortus development with IC50 values of 2.8–5.6 μM. Circular dichroism spectropolarimetry indicated that the ponericins are unstructured in aqueous solution but adopt α-helical conformations in lipid mimetic environments. We show that the ponericins induce non-specific membrane perturbation, which confers broad-spectrum antimicrobial, insecticidal, cytotoxic, hemolytic, and algogenic activities, with activity across all assays typically correlated. We also show for the first time that ponericins induce spontaneous pain behaviour when injected in mice. We propose that the broad-spectrum activity of the ponericins enables them to play both a predatory and defensive role in neoponeran ants, consistent with their high abundance in venom. This study reveals a broader functionality for ponericins than previously assumed, and highlights both the opportunities and challenges in pursuing ant venom peptides as potential therapeutics.

Modelling the mechanism and kinetics of the radical scavenging activity of iminostilbene

Iminostilbene (IS) (5H‐dibenz[b,f]azepine) is frequently used as a polymerization inhibitor to stop the reactions of radical polymerization. The compound itself is considered an antioxidant; however, little is known about the mechanism and kinetics of its activity. In this study, the antiradical activity of IS against typical radicals i.e. HO•, HOO• and TBO• (tert-butoxy radical) has been evaluated by computational methods. It was found that IS exhibits excellent HO• and TBO• scavenging activity in gas phase and in lipid-mimetic environment (benzene), whereas its activity against HOO• is moderate with koverall = ~ 104 M−1 s−1. The overall rate constants of the HO• antiradical activity are koverall = 7.06 × 1011 and 7.20 × 109 M−1 s−1 in gas phase and benzene, respectively, whereas for the TBO• antiradical activity in benzene it is 2.10 × 108 M−1 s−1. Comparison with the experimentally determined rate constant in benzene (k(BTO•) = 7.8 × 108 M−1s−1) yields the kcalculated/kexperimetal ratio of 0.3; thus, the computed kinetic data are reasonably accurate. It was shown that the TBO• and HOO• radicals scavenging of IS mostly follows the FHT mechanism at the NH bond; however, in case of the HO• radical both FHT (at NH bond) and RAF (at C10 position) mechanisms contribute to the observable activity.

Is Indolinonic Hydroxylamine a Promising Artificial Antioxidant?

Indolinonic hydroxylamine (IH) is a new-generation artificial antioxidant that, due to its ability to fractionate into apolar environments, is considered for prevention against lipid peroxidation. For this reason, it is important to understand, and compare, its activity in polar and nonpolar environments. In this study, the antioxidant activity of IH has been evaluated against HO• and HOO• radicals in water and, for a lipid-mimetic environment, pentyl ethanoate solvent, using kinetic calculations. It was found that the overall reaction rate constant of the HO• radical scavenging is more than 7 times higher in aqueous (8.98 × 109 M-1 s-1) than in apolar (1.22 × 109 M-1 s-1) media. However, HOO• scavenging was 35 times faster in apolar media (1.00 × 105 M-1 s-1 vs 2.80 × 103 M-1 s-1). In a lipid environment, the HAT mechanism was favored for the antioxidant activity for both radical species, whereas in aqueous solution the SET mechanism defined the HO• scavenging, while HAT described the HOO• scavenging. IH was shown to be one of the most active antioxidants in lipid environment, an essential characteristic for the protection of biological systems.

Protein Misfolding in Lipid-Mimetic Environments.

Among various cellular factors contributing to protein misfolding and subsequent aggregation, membranes occupy a special position due to the two-way relations between the aggregating proteins and cell membranes. On one hand, the unstable, toxic pre-fibrillar aggregates may interact with cell membranes, impairing their functions, altering ion distribution across the membranes, and possibly forming non-specific membrane pores. On the other hand, membranes, too, can modify structures of many proteins and affect the misfolding and aggregation of amyloidogenic proteins. The effects of membranes on protein structure and aggregation can be described in terms of the "membrane field" that takes into account both the negative electrostatic potential of the membrane surface and the local decrease in the dielectric constant. Water-alcohol (or other organic solvent) mixtures at moderately low pH are used as model systems to study the joint action of the local decrease of pH and dielectric constant near the membrane surface on the structure and aggregation of proteins. This chapter describes general mechanisms of structural changes of proteins in such model environments and provides examples of various proteins aggregating in the "membrane field" or in lipid-mimetic environments.

On the Relative Stability of Dimeric Interfaces of the Mu-Opioid Receptor Inferred from Recent Crystallographic Studies

The mu-opioid (MOP) receptor is the predominant target for most clinically used analgesics. In response to demonstrated correlations between opioid dependence and the MOP receptor, researchers have focused on alternative targets. Opioid receptor oligomers are among those that have been suggested to mediate analgesia without the common opioid-related adverse effects. Thus, obtaining a molecular-level understanding of the nature of receptor-receptor interactions in the membrane, either within, or between receptor subtypes, can both create new opportunities for drug discovery, and address the role of oligomerization in receptor function. The recent MOP receptor crystal structure has inspired hypotheses of dimerization contacts, specifically: a closely packed interface involving transmembrane (TM) helices TM5 and TM6, and a less compact one involving TM1, TM2, and helix 8 (H8). These interfaces exhibit similar arrangements to those found in crystals of the chemokine receptor CXCR4 and the kappa-opioid receptor, respectively. While it is tempting to speculate that the tighter TM5/TM6 arrangement has physiological relevance, additional studies are necessary to a) understand the relative dimer stability at TM5/TM6, TM1/TM2/H8, and other interfaces in the membrane, b) evaluate the contribution of the engineered T4 lysozyme (T4L) to the TM5/TM6 association, and c) investigate possible variability across different receptors. To begin to address these questions we have performed umbrella sampling molecular dynamics simulations of coarse-grained representations of the MOP receptor interacting at the TM5/TM6 (with and without T4L) or TM1/TM2/H8 interfaces in an explicit lipid-water environment. We have derived relative estimates of the dimerization free energy at the two specific interfaces and from these we suggest relative dimer lifetimes and dimeric fractions in a lipid mimetic environment. This information can help design future experiments aimed at understanding the role of dimerization in receptor function.

Dissecting the Roles of the Extracellular Regions in Apelin Peptide-Receptor Binding

The apelin receptor is a class A G-protein coupled receptor with homology to the angiotensin II receptor and the CXCR4 and CCR5 chemokine receptors. It is expressed at high levels in lung, heart, adipose tissue, kidney, spleen and in some areas of the brain. The apelin receptor gene is also upregulated and overexpressed in one-third of human tumours. The receptor is assumed to consist of a transmembrane region with 7 helical segments, an intracellular and an extracellular domain. The extracellular domain, made up of the N-terminal tail and three extracellular loops (EL1, 2 and 3), exhibits low sequence homology to other GPCRs. Several somatic mutations in loops have been linked to diseases, pointing out the importance of loops in the activation and/or binding of GPCRs to their cognate ligands. We carried out a physicochemical characterization of the N-terminus and extracellular loops of the apelin receptor in lipid mimetic environments. The N-terminus (attached to the first transmembrane domain) and EL2 were produced as recombinant proteins while the EL peptides and a fluorescently tagged apelin were synthesized by solid-phase peptide synthesis. All species were purified by high-performance liquid chromatography. Characterization both of the peptides in isolation and of the binding between apelin and extracellular loop peptides was carried out using circular dichroism spectroscopy, fluorescence resonance energy transfer and nuclear magnetic resonance spectroscopy. These results provide insight into understanding the role of the each of the extracellular regions of this receptor at the molecular level and constitute important information the apelin receptor for the development of therapeutics targeting this system.

Probing Binding of Apelin to the Extracellular Loops of the Apelin Receptor (APJ) in Lipid Mimetic Environments

a Department of Biochemistry & Molecular Biology and b Department of Chemistry, Dalhousie University, Halifax, NS B3H 1X5.The apelin receptor, previously called APJ, is a G protein-coupled receptor highly expressed in the central nervous system (CNS), cardiovascular system, the adipoinsular axis and the mammary glands, among other tissues and organ systems. The apelinergic system plays important biological functions in the regulation of blood pressure, blood glucose, drinking behavior and food intake. The action of this system is also implicated in tumour angiogenesis, diabetes and cardiovascular diseases. In addition, the apelin receptor is a co-receptor for human immunodeficiency virus type 1 (HIV1) and simian immunodeficiency virus (SIV). Despite these roles, the mechanism of activation of apelin receptor by its cognate ligand has not been studied at the molecular level. Following the “divide and conquer” approach for membrane protein characterization, we present the biophysical characterization of the interaction of two extracellular loops of the apelin receptor (extracellular loops 1, EL1 and 3, EL3) with a fluorescently-tagged apelin analogue in lipid environment. Peptides were synthesized by solid phase peptide synthesis and purified by high-performance liquid chromatography. Characterization both of the peptides in isolation and of the binding between apelin and EL peptides is provided using circular dichroism spectroscopy, fluorescence resonance energy transfer and nuclear magnetic resonance spectroscopy. These results provide insight into understanding of the apelinergic system at the molecular level and provide the first structural information on the apelin receptor for the development of therapeutics targeting this system.

Structure and Function of the Influenza A M2 Proton Channel

The M2 protein of influenza A viruses forms a tetrameric pH-activated proton-selective channel that is targeted by the amantadine class of antiviral drugs. Its ion channel function has been extensively studied by electrophysiology and mutagenesis; however, the molecular mechanism of proton transport is still elusive, and the mechanism of inhibition by amantadine is controversial. We review the functional data on proton channel activity, molecular dynamics simulations of the proton conduction mechanism, and high-resolution structural and dynamical information of this membrane protein in lipid bilayers and lipid-mimetic detergents. These studies indicate that elucidation of the structural basis of M2 channel activity and inhibition requires thorough examination of the complex dynamics and conformational plasticity of the protein in different lipid bilayers and lipid-mimetic environments.

Correlation of Three-dimensional Structures with the Antibacterial Activity of a Group of Peptides Designed Based on a Nontoxic Bacterial Membrane Anchor

To understand the functional differences between a nontoxic membrane anchor corresponding to the N-terminal sequence of the Escherichia coli enzyme IIA(Glc) and a toxic antimicrobial peptide aurein 1.2 of similar sequence, a series of peptides was designed to bridge the gap between them. An alteration of a single residue of the membrane anchor converted it into an antibacterial peptide. Circular dichroism spectra indicate that all peptides are disordered in water but helical in micelles. Structures of the peptides were determined in membrane-mimetic micelles by solution NMR spectroscopy. The quality of the distance-based structures was improved by including backbone angle restraints derived from a set of chemical shifts ((1)H(alpha), (15)N, (13)C(alpha), and (13)C(beta)) from natural abundance two-dimensional heteronuclear correlated spectroscopy. Different from the membrane anchor, antibacterial peptides possess a broader and longer hydrophobic surface, allowing a deeper penetration into the membrane, as supported by intermolecular nuclear Overhauser effect cross-peaks between the peptide and short chain dioctanoyl phosphatidylglycerol. An attempt was made to correlate the NMR structures of these peptides with their antibacterial activity. The activity of this group of peptides does not correlate exactly with helicity, amphipathicity, charge, the number of charges, the size of the hydrophobic surface, or hydrophobic transfer free energy. However, a correlation is established between the peptide activity and membrane perturbation potential, which is defined by interfacial hydrophobic patches and basic residues in the case of cationic peptides. Indeed, (31)P solid state NMR spectroscopy of lipid bilayers showed that the extent of lipid vesicle disruption by these peptides is proportional to their membrane perturbation potential.

Secondary structure of human apolipoprotein A-I(1–186) in lipid-mimetic solution

The solution structure of an apoA-I deletion mutant, apoA-I(1–186) was determined by the chemical shift index (CSI) method and the torsion angle likelihood obtained from shift and sequence similarity (TALOS) method, using heteronuclear multidimensional NMR spectra of [u- 13C, u- 15N, u-50% 2H]apoA-I(1–186) in the presence of sodium dodecyl sulfate (SDS). The backbone resonances were assigned from a combination of triple-resonance data (HNCO, HNCA, HN(CO)CA, HN(CA)CO and HN(COCA)HA), and intraresidue and sequential NOEs (three-dimensional (3D) and four-dimensional (4D) 13C- and 15N-edited NOESY). Analysis of the NOEs, H α, C α and C′ chemical shifts shows that apoA-I(1–186) in lipid-mimetic solution is composed of α-helices (which include the residues 8–32, 45–64, 67–77, 83–87, 90–97, 100–140, 146–162, and 166–181), interrupted by short irregular segments. There is one relatively long, irregular and mostly flexible region (residues 33–44), that separates the N-terminal domain (residues 1–32) from the main body of protein. In addition, we report, for the first time, the structure of the N-terminal domain of apoA-I in a lipid-mimetic environment. Its structure (α-helix 8–32 and flexible linker 33–44) would suggest that this domain is structurally, and possibly functionally, separated from the other part of the molecule.

Conformation of human apolipoprotein C-I in a lipid-mimetic environment determined by CD and NMR spectroscopy.

The high-resolution conformation of human apoC-I in complexes with sodium dodecyl sulfate (SDS) is presented. As estimated from CD data, apoC-I adopts 54% helical secondary structure when bound to SDS, which is similar to the helical content previously found with phospholipids. The NMR-derived conformation of apoC-I is composed of two amphipathic helices, residues 7-29 and 38-52, separated by a flexible linker. The N-terminal helix contains a mobile hinge involving residues 12-15. The hydrophobic side chains cluster on the nonpolar face of both helices, thus forming two discrete lipid-binding sites in the N-terminal helix and one in the C-terminal helix. As suggested by amide proton resonance line widths and deuterium exchange rates, the N-terminal helix is more flexible and may bind less tightly to the detergent than the C-terminal helix. The different mobility of both helices appears to be related to side-chain composition, rather than length of the amphipathic helix, and may play a role in the function of apoC-I as an activator of lecithin:cholesterol acyltransferase (LCAT). A model is suggested in which the C-terminal helix serves as a lipid anchor while the N-terminal helix may hinge off the lipid surface to make specific contacts with LCAT.

Rapid release and unusual stability of immunodominant peptide 45-89 from citrullinated myelin basic protein.

Myelin basic protein (MBP) exists in a population of isoforms and isomers. The 18.5 kDa MBP-C1, the main human adult isoform, has 170 residues and is relatively unmodified, whereas the same isoform can be citrullinated on six arginine residues to create the MBP-C8 (MBP Cit6) isomer. MBP Cit6 dominates in MS brain, accounting for 45% rather than 25% of the population of MBP isomers. In the fulminant form of MS, known as Marburg's Disease, 18 of the 19 arginines in MBP are citrullinated (MBP Cit18). Citrullination of MBP could lead to instability of myelin or limited remyelination. In this investigation, the susceptibilities to degradation by cathepsin D of MBP Cit6 and MBP-C1, both from normal and MS brain tissue, and Marburg MBP Cit18 were compared. The pattern of digestion was similar, and no differences of corresponding isomers in normal and MS brain were noted. However, normal MBP Cit6 was degraded 10-fold more rapidly than MBP-C1, and MBP Cit18 was degraded even more rapidly. MBP peptide 45-89 was preserved regardless of isomer type or source. Its generation was directly related to the citrulline content of the MBP substrate being 4 times faster in normal MBP Cit6 and 35 times faster in Marburg MBP Cit18 than in normal MBP-C1. Peptide 45-89 from a citrullinated MBP exhibited more deamidation, and, regardless of source, showed an alpha-helix structure in a lipid mimetic environment. We postulate that the generation of MBP peptides, including those that are dominant and encephalitogenic, is directly related to deimination of arginine to citrulline in MBP.

Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E

The N-terminal domain of human apolipoprotein E3 (apoE3) adopts an elongated, globular four helix bundle conformation in the lipid-free state. Upon lipid binding, the protein is thought to undergo a significant conformational change that is essential for manifestation of its low density lipoprotein receptor recognition properties. We have used fluorescence resonance energy transfer (FRET) to characterize helix repositioning which accompanies lipid interaction of this protein. ApoE3(1-183) possesses a single cysteine at position 112 and four tryptophan residues (positions 20, 26, 34, and 39). Modification of Cys112 with the chromophore, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS) was specific and did not alter the secondary structure content of the protein. The efficiency of energy transfer from donor Trp residues to the AEDANS moiety was 49% in buffer, consistent with close proximity of the chromophores. Guanidine HCl titration experiments induced characteristic changes in the efficiency of energy transfer, indicating that FRET data faithfully reports on the conformational status of the protein. Interaction of AEDANS-apoE3(1-183) with dimyristoylphosphatidylcholine to form disk particles, or with detergent micelles, resulted in large decreases in the efficiency of energy transfer. Distance calculations based on the FRET measurements revealed that lipid binding increases the average distance between the four Trp donors and the AEDANS acceptor from 23 A to 44 A. The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable apolipoproteins and are consistent with the hypothesis that, upon lipid binding, apoE3(1-183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation.

The helix-hinge-helix structural motif in human apolipoprotein A-I determined by NMR spectroscopy.

The conformation of a synthetic peptide of 46 residues from apoA-I was investigated by fluorescence, CD, and 2D NMR spectroscopies in lipid-mimetic environments. ApoA-I(142-187) is mainly unstructured in water but helical in SDS or dodecylphosphocholine (DPC), although the peptide only associates with DPC at approximately the critical micellar concentration. Solution structures of apoA-I(142-187) were determined by distance geometry calculations based on 450 (in DPC-d38) or 397 (in SDS-d25) NOE-derived distance restraints, respectively. Backbone RMSDs for superimposing the two helical regions 146-162 and 168-182 are 0.98 +/- 0.22 (2.38 +/- 0.20) and 1.99 +/- 0.42 (2.02 +/- 0.21) A in DPC (SDS), respectively. No interhelical NOE was found, suggesting that helix-helix interactions between the two helical domains in apoA-I(142-187) are unlikely. Similar average, curved helix-hinge-helix structures were found in both SDS and DPC micelles with the hydrophobic residues occupying the concave face, indicating that hydrophobic interactions dominate. Intermolecular NOESY experiments, performed in the presence of 50% protonated SDS, confirm that the two amphipathic helices and Y166 in the hinge all interact with the micelle. The involvement of Y166 in lipid binding is supported by fluorescence spectroscopy as well. On the basis of all the data above, we propose a model for the peptide-lipid complexes wherein the curved amphipathic helix-hinge-helix structural motif straddles the micelle. The peptide-aided signal assignment achieved for apoA-I(122-187) (66mer) and apoA-I suggests that such a structural motif is retained in the longer peptide and most likely in the intact protein.

Structure and dynamics of detergent-solubilized M13 coat protein (an integral membrane protein) determined by 13C and 15N nuclear magnetic resonance spectroscopy

The major coat protein of the filamentous bacteriophage M13 is inserted as an integral protein in the inner membrane of the Escherichia coli host upon infection. M13 coat protein is an ideal model membrane protein and has been the target of many biophysical studies. An overview is presented here of the application of nuclear magnetic resonance spectroscopy to the study of the structure and dynamics of M13 coat protein in several lipid-mimetic environments. The coat protein may be biosynthetically enriched with 13C- and 15N-labelled amino acids, allowing the resolution and assignment of individual nuclei. Structural fluctuations at selected sites have been monitored using 13C relaxation and isotope-detected amide hydrogen exchange kinetics. A model is proposed for the structure of a coat protein dimer in detergent micelles.