High-resolution solution structure of siamycin II: Novel amphipathic character of a 21-residue peptide that inhibits HIV fusion

Summary The 21-amino acid peptides siamycin II (BMY-29303) and siamycin I (BMY-29304), derived from Streptomyces strains AA3891 and AA6532, respectively, have been found to inhibit HIV-1 fusion and viral replication in cell culture. The primary sequence of siamycin II is CLGIGSCNDFAGCGYAIVCFW. Siamycin I differs by only one amino acid; it has a valine residue at position 4. In both peptides, disulfide bonds link Cys¹ with Cys¹³ and Cys⁷ with Cys¹⁹, and the side chain of Asp⁹ forms an amide bond with the N-terminus. Siamycin II, when dissolved in a 50:50 mixture of DMSO and H₂O, yields NOESY spectra with exceptional numbers of cross peaks for a peptide of this size. We have used 335 NOE distance constraints and 13 dihedral angle constraints to generate an ensemble of 30 siamycin II structures; these have average backbone atom and all heavy atom rmsd values to the mean coordinates of 0.24 and 0.52 Å, respectively. The peptide displays an unusual wedge-shaped structure, with one face being predominantly hydrophobic and the other being predominantly hydrophilic. Chemical shift and NOE data show that the siamycin I structure is essentially identical to siamycin II. These peptides may act by preventing oligomerization of the HIV transmembrane glycoprotein gp41, or by interfering with interactions between gp41 and the envelope glycoprotein gp120, the cell membrane or membrane-bound proteins [Frèchet, D. et al. (1994) Biochemistry, 33, 42–50]. The amphipathic nature of siamycin II and siamycin I suggests that a polar (or apolar) site on the target protein may be masked by the apolar (or polar) face of the peptide upon peptide/protein complexation.Abbreviations ABNR adopted basis Newton Raphson - AIDS acquired immunodeficiency syndrome - CW continuous wave - DMSO dimethylsulfoxide - DQF-COSY two-dimensional double-quantum-filtered correlation spectroscopy - HIV human immunodeficiency virus - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - ppm parts per million - P.E.-COSY two-dimensional primitive exclusive correlation spectroscopy - REDAC redundant dihedral angle constraint - rf radio frequency - rmsd root-mean-square difference - SIV simian immunodeficiency virus - sw spectral width - _m mixing time - TOCSY two-dimensional total correlation spectroscopy - TSP trimethylsilyl-2,2,3,3-²H₄-propionate - 2D two-dimensional     

Membrane Association via an Amino-terminal Amphipathic Helix Is Required for the Cellular Organization and Function of RNase II

The subcellular localization of the exoribonuclease RNase II is not known despite the advanced biochemical characterization of the enzyme. Here we report that RNase II is organized into cellular structures that appear to coil around the Escherichia coli cell periphery and that RNase II is associated with the cytoplasmic membrane by its amino-terminal amphipathic helix. The helix also acts as an autonomous transplantable membrane binding domain capable of directing normally cytoplasmic proteins to the membrane. Assembly of the organized cellular structures of RNase II required the RNase II amphipathic membrane binding domain. Co-immunoprecipitation of the protein from cell extracts indicated that RNase II interacts with itself. The RNase II self-interaction and the ability of the protein to assemble into organized cellular structures required the membrane binding domain. The ability of RNase II to maintain cell viability in the absence of the exoribonuclease polynucleotide phosphorylase was markedly diminished when the RNase II cellular structures were lost due to changes in the amphipathicity of the amino-terminal helix, suggesting that membrane association and assembly of RNase II into organized cellular structures play an important role in the normal function of the protein within the bacterial cell.     

Lipid-induced Pore Formation of the Bacillus thuringiensis Cry1Aa Insecticidal Toxin

After activation, Bacillus thuringiensis (Bt) insecticidal toxin forms pores in larval midgut epithelial cell membranes, leading to host death. Although the crystal structure of the soluble form of Cry1Aa has been determined, the conformation of the pores and the mechanism of toxin interaction with and insertion into membranes are still not clear. Here we show that Cry1Aa spontaneously inserts into lipid mono- and bilayer membranes of appropriate compositions. Fourier Transform InfraRed spectroscopy (FTIR) indicates that insertion is accompanied by conformational changes characterized mainly by an unfolding of the β-sheet domains. Moreover, Atomic Force Microscopy (AFM) imaging strongly suggests that the pores are composed of four subunits surrounding a 1.5 nm diameter central depression. Received: 14 July 2000/Revised: 28 December 2000     

On the structure and function of apolipoproteins: more than a family of lipid-binding proteins

Exchangeable apolipoproteins have been the subject of intense biomedical investigation for decades. However, only in recent years the elucidation of the three-dimensional structure reported for several members of the apolipoprotein family has provided insights into their functions at a molecular level for the first time. Moreover, the role of exchangeable apolipoproteins in several cellular events distinct from lipid metabolism has recently been described. This review summarizes these contributions, which have not only allowed the identification of the apolipoprotein domains that determine substrate binding specificity and/or affinity but also the plausible molecular mechanism(s) involved.     

The SU Glycoprotein 120 from HIV-1 Penetrates into Lipid Monolayers Mimicking Plasma Membranes

Increasing evidence suggests that the HIV envelope binds through its surface (SU) gp120 not only to receptors and coreceptors, but also to other components of the cellular membrane where the glycolipids appear to be good candidates. To assess the ability of HIV-1 SU gp120 to penetrate into phospholipid membranes, we carried out a study of the interactions between a recombinant SU gp120 from HIV-1/HXB2 and artificial lipid monolayers mimicking the composition of the outer leaflet of the lymphocytes and which were spread at the air-water interface. We show that the protein, in its aggregated form, has amphipathic properties and that the insertion of this amphipathic species into lipids is favored by the presence of sphingomyelin. Furthermore, cholesterol enhances the penetration into mixed phosphatidylcholine-sphingomyelin monolayers. Coexistence of different physical states of the lipids and thus of domains appears to play a major role for protein penetration independently of the presence of receptors and coreceptors. Received: 24 April 2000/Revised: 11 July 2000     

Membrane Curvature Induction and Tubulation Are Common Features of Synucleins and Apolipoproteins

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.     

Mechanistic aspects of CPP-mediated intracellular drug delivery: Relevance of CPP self-assembly

In recent years, cell-penetrating peptides have proven to be an efficient intracellular delivery system. The mechanism for CPP internalisation, which first involves interaction with the extracellular matrix, is followed in most cases by endocytosis and finally, depending on the type of endocytosis, an intracellular fate is reached. Delivery of cargo attached to a CPP requires endosomal release, for which different methods have recently been proposed. Positively charged amino acids, hydrophobicity and/or amphipathicity are common to CPPs. Moreover, some CPPs can self-assemble. Herein is discussed the role of self assembly in the cellular uptake of CPPs. Sweet Arrow Peptide (SAP) CPP has been shown to aggregate by CD and TEM (freeze-fixation/freeze-drying), although the internalised species have yet to be identified as either the monomer or an aggregate.     

Fungicidal effect and the mode of action of piscidin 2 derived from hybrid striped bass

Piscidin 2 (P2), a 22-residue cationic peptide isolated from the mast cells of hybrid striped bass, has potent antibacterial activities. However, its antifungal properties are not completely understood. In the current study, we investigated the antifungal effects and mode of action of P2. P2 exhibited potent antifungal activity against human pathogenic fungi. To understand the fungicidal properties of P2, we focused on a membrane-active mechanism of the peptide by in vivo and in vitro testing. Flow cytometric analysis using bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and protoplast regeneration experiments showed that P2 caused fungal membrane damage. Furthermore, fluorescence analysis using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed that P2 created pores in fungal membranes. These results were confirmed with dye leakage tests by using liposomes composed of phosphatidylcholine/phosphatidylserine (3:1, w/w), which mimicked fungal membranes. The present study indicated that P2 exerts its fungicidal effects by perturbing membrane activities.     

Selective amphipathic nature of chlorpromazine binding to plasma membrane bilayers

Chlorpromazine (CPZ), an antipsychotic agent shown to inhibit the action of various neurophysiological receptors, also exhibits preferential association with the plasma membrane, inducing stomatocytic morphological response in red blood cells (RBC). Given the cationic nature of CPZ, fluorimetry, pH titration, and red cell morphological studies were performed to assess the associative predilection of CPZ for anionic membrane components. CPZ fluorescence intensity increased 320-370% upon addition of phosphatidylcholine (PC) small unilamellar vesicles (SUVs) to aqueous CPZ, indicating an affinity of the drug for lipidic phases. After removal of unbound drug, CPZ fluorescence increased up to 92% with increasing phosphatidylserine (PS) in the lipid phase (up to 30 mol% of total lipid), suggesting a preferential association of the drug with anionic lipids. In studies of pH titration, the pK_a of CPZ in the presence of Triton X-100 micelles or phospholipid SUVs increased with increasing anionicity of the lipidic phase [7.8 with Triton X-100, 8.0 with PC, 8.3 with phosphatidylglycerol (PG)], lending further support to preferential drug interaction with anionic lipidic components. At 0 °C, CPZ-induced red cell shape change was less extensive in cells made echinocytic by adenosine triphosphate (ATP) depletion, compared to cells made echinocytic by PS treatment following vanadate preincubation. This suggests that polyphosphoinositide lipids are CPZ membrane binding sites. Since polyphosphoinositide lipids are implicated as important intermediates in a number of receptor-mediated cell signaling pathways, evidence of association with these specific lipids provides a means by which psychoactive drugs may induce neurophysiological effects through direct interaction with general membrane components.     

Design and synthesis of cell selective α/β-diastereomeric peptidomimetic with potent in vivo antibacterial activity against methicillin resistant S. Aureus

Design of therapeutically viable antimicrobial peptides with cell selectivity against microorganisms is an important step towards the development of new antimicrobial agents. Here, we report four de novo designed, short amphipathic sequences based on a α-helical template comprising of Lys, Trp and Leu or their corresponding D-and/or β-amino acids. Sequence A-12 was protease susceptible whereas its α/β-diastereomeric analogue UNA-12 was resistant to trypsin and proteinase K up to 24 h. A-12 and UNA-12 exhibited broad-spectrum antibacterial activity (MIC: 2–32 µg/mL) against pathogens including methicillin resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE). Interestingly, A-12 was found to be most toxic (>50% haemolytic at 250 µg/mL) whereas UNA-12 was found to be non cytotoxic among the all analogues against hRBCs and human keratinocytes. Interaction studies with artificial membranes by tryptophan fluorescence and acrylamide quenching assay demonstrated A-12 interacted equally in bacterial as well as mammalian mimic membrane whereas UNA-12 was found to be more selective towards bacterial mimic membrane. Further microscopic tool has revealed membrane damaging ability of A-12 and UNA-12 with bactericidal mode of action against MRSA. Encouragingly, peptidomimetics analogue UNA-12 showed remarkable safety and efficacy against MRSA in in-vivo neutropenic mice thigh infection model. In summary, simultaneous replacement of the natural amino acids with D-/β-congeners is a promising strategy for designing of potent, cell selective and protease stable peptide based antibiotics.