Influenza A virus protein PB1-F2: synthesis and characterization of the biologically active full length protein and related peptides.

Recently the discovery of a novel 87 amino acid influenza A virus (IAV) protein, named PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most of the known human IAV isolates. Using optimized protocols, full length biologically active sPB1-F2 and a number of fragments have been synthesized by following either the standard elongation SPPS method or by native chemical ligation of unprotected N- and C-terminal peptide fragments at the histidine and cysteine residues located in position 41 and 42 of the native sequence, respectively. The ligation procedure afforded the most efficient synthesis of sPB1-F2 and facilitated the generation of various mutants of sPB1-F2 from pre-synthesized peptide fragments. During the synthesis of sPB1-F2, the formation of succinimide and subsequent conversion to the piperidine derivative at the aspartic acid residue in position 23 was observed. This reaction was forestalled by applying specific modifications to the SPPS protocol. The chain-elongation SPPS protocol is optimal for producing small peptides of sPB1-F2, their derivatives and precursors for a subsequent ligation protocol, while the full length protein, mutants and labelled derivatives are more conveniently and efficiently synthesized by SPPS protocols that include native chemical ligation. The molecular identity of sPB1-F2 was confirmed by peptide mapping, mass spectrometry, N-terminal sequencing, (1)H NMR spectroscopy and Western blot analysis. The latter analysis afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization, a phenomenon observed both for full length sPB1-F2 and fragments thereof, as well as for its full length viral counterpart. Our synthesis protocols open the field for multiple biological and structural studies on sPB1-F2 that, similar to the molecule expressed in an IAV context, induces apoptosis and interacts with membranes in vitro and in vivo, as shown in previous studies.     

Development of potent anti-infective agents from Silurana tropicalis: Conformational analysis of the amphipathic,alpha-helical antimicrobial peptide XT-7 and its non-haemolytic analogue [G4K]XT-7

Peptide XT-7 (GLLGP⁵LLKIA¹⁰AKVGS¹⁵NLL.NH₂) is a cationic, leucine-rich peptide, first isolated from skin secretions of the frog, Silurana tropicalis (Pipidae). The peptide shows potent, broad-spectrum antimicrobial activity but its therapeutic potential is limited by haemolytic activity (LC₅₀ = 140 µM). The analogue [G4K]XT-7, however, retains potent antimicrobial activity but is non-haemolytic (LC₅₀ > 500 µM). In order to elucidate the molecular basis for this difference in properties, the three dimensional structures of XT-7 and the analogue have been investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, both peptides lack secondary structure. In a 2,2,2-trifluoroethanol (TFE-d₃)-H₂O mixed solvent system, XT-7 is characterised by a right handed α-helical conformation between residues Leu³ and Leu¹⁷ whereas [G4K]XT-7 adopts a more restricted α-helical conformation between residues Leu⁶ and Leu¹⁷. A similar conformation for XT-7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellular media was observed with a helical segment between Leu³ and Leu¹⁷. However, differences in side chain orientations restricting the hydrophilic residues to a smaller patch resulted in an increased hydrophobic surface relative to the conformation in TFE-H₂O. Molecular modelling of the structures obtained in our study demonstrates the amphipathic character of the helical segments. It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly⁴ → Lys in XT-7 arises from a decrease in both helicity and hydrophobicity. These studies may facilitate the development of potent but non-toxic anti-infective agents based upon the structure of XT-7.     

Interaction of local anesthetics with a peptide encompassing the IV/S4-S5 linker of the Na+ channel

The peptide pIV/S4-S5 encompasses the cytoplasmic linker between helices S4-S5 in domain IV of the voltage-gated Na+ channel, residues 1644-1664. The interaction of two local anesthetics (LA), lidocaine and benzocaine, with pIV/S4-S5 has been studied by DOSY, heteronuclear NMR 1H-15N-HSQC spectroscopy and computational methods. DOSY indicates that benzocaine, a neutral ester, exhibits stronger interaction with pIV/S4-S5 than lidocaine, a charged amine-amide. Weighted average chemical shifts, Deltadelta(1H-15N), show that benzocaine affects residues L1653, M1655 and S1656 while lidocaine slightly perturbs residues I1646, L1649 and A1659, L1660, near the N- and C-terminus, respectively. Computational methods confirmed the stability of the benzocaine binding and the existence of two binding sites for lidocaine. Even considering that the approach of studying the peptide in the presence of a co-solvent (TFE/H2O, 30%/70% v/v) has an inherently limited implication, our data strongly support the existence of multiple LA binding sites in the IV/S4-S5 linker, as suggested in the literature. In addition, we consider that LA can bind to the S4-S5 linker with diverse binding modes and strength since this linker is part of the receptor for the "inactivation gate particle". Conditions for devising new functional studies, aiming to better understand Na+ channel functionality as well as the various facets of LA pharmacological activity are proposed in this work.     

Structure and Topology of the Huntingtin 1–17 Membrane Anchor by a Combined Solution and Solid-State NMR Approach

The very amino-terminal domain of the huntingtin protein is directly located upstream of the protein’s polyglutamine tract, plays a decisive role in several important properties of this large protein and in the development of Huntington’s disease. This huntingtin 1–17 domain is on the one hand known to markedly increase polyglutamine aggregation rates and on the other hand has been shown to be involved in cellular membrane interactions. Here, we determined the high-resolution structure of huntingtin 1–17 in dodecyl phosphocholine micelles and the topology of its helical domain in oriented phosphatidylcholine bilayers. Using two-dimensional solution NMR spectroscopy the low-energy conformations of the polypeptide were identified in the presence of dodecyl phosphocholine detergent micelles. In a next step a set of four solid-state NMR angular restraints was obtained from huntingtin 1–17 labeled with ¹⁵N and ²H at selected sites. Of the micellar ensemble of helical conformations only a limited set agrees in quantitative detail with the solid-state angular restraints of huntingtin 1–17 obtained in supported planar lipid bilayers. Thereby, the solid-state NMR data were used to further refine the domain structure in phospholipid bilayers. At the same time its membrane topology was determined and different motional regimes of this membrane-associated domain were explored. The pronounced structural transitions of huntingtin 1–17 upon membrane-association result in a α-helical conformation from K6 to F17, i.e., up to the very start of the polyglutamine tract. This amphipathic helix is aligned nearly parallel to the membrane surface (tilt angle ∼77°) and is characterized by a hydrophobic ridge on one side and an alternation of cationic and anionic residues that run along the hydrophilic face of the helix. This arrangement facilitates electrostatic interactions between huntingtin 1–17 domains and possibly with the proximal polyglutamine tract.     

Structural characterization of the transmembrane proximal region of the hepatitis C virus E1 glycoprotein

A detailed knowledge of the mechanism of virus entry represents one of the most promising approaches to develop new therapeutic strategies. However, viral fusion is a very complex process involving fusion glycoproteins present on the viral envelope. In the two hepatitis C virus envelope proteins, E1 and E2, several membranotropic regions with a potential role in the fusion process have been identified. Among these, we have selected the 314-342 E1 region. Circular Dichroism data indicate that the peptide exhibits a clear propensity to adopt a helical folding in different membrane mimicking media, such as mixtures of water with fluorinated alcohols and phospholipids, with a slight preference for negative charged bilayers. The 3D structure of E1_314-342 peptide, calculated by 2D-NMR in a low-polarity environment, consists of two helical stretches encompassing residues 319-323 and 329-338 respectively. The peptide, presenting a largely apolar character, interacts with liposomes, as indicated by fluorescence and electron spin resonance spectra. The strength of the interaction and the deepness of peptide insertion in the phospholipid membrane are modulated by the bilayer composition, the interaction with anionic phospholipids being among the strongest ever observed. The presence of cholesterol also affects the peptide-bilayer interaction, favoring the peptide positioning close to the bilayer surface. Overall, the experimental data support the idea that this region of E1 might be involved in membrane destabilization and viral fusion; therefore it may represent a good target to develop anti-viral molecules.     

NMR structures of the histidine-rich peptide LAH4 in micellar environments: membrane insertion, pH-dependent mode of antimicrobial action, and DNA transfection

The LAH4 family of histidine-rich peptides exhibits potent antimicrobial and DNA transfection activities, both of which require interactions with cellular membranes. The bilayer association of the peptides has been shown to be strongly pH-dependent, with in-planar alignments under acidic conditions and transmembrane orientations when the histidines are discharged. Therefore, we investigated the pH- and temperature-dependent conformations of LAH4 in DPC micellar solutions and in a TFE/PBS solvent mixture. In the presence of detergent and at pH 4.1, LAH4 adopts helical conformations between residues 9 and 24 concomitantly with a high hydrophobic moment. At pH 6.1, a helix-loop-helix structure forms with a hinge encompassing residues His¹⁰-Ala¹³. The data suggest that the high density of histidine residues and the resulting electrostatic repulsion lead to both a decrease in the pK values of the histidines and a less stable α-helical conformation of this region. The hinged structure at pH 6.1 facilitates membrane anchoring and insertion. At pH 7.8, the histidines are uncharged and an extended helical conformation including residues 4-21 is again obtained. LAH4 thus exhibits a high degree of conformational plasticity. The structures provide a stroboscopic view of the conformational changes that occur during membrane insertion, and are discussed in the context of antimicrobial activity and DNA transfection.     

The solution structure of an N-terminally truncated version of the yeast CDC24p PB1 domain shows a different beta-sheet topology

Phox and Bem1 (PB1) domains mediate protein-protein interactions via the formation of homo- or hetero-dimers. The C-terminal PB1 domain of yeast cell division cycle 24 (CDC24p), a guanine-nucleotide exchange factor involved in cell polarity establishment, is known to interact with the PB1 domain occurring in bud emergence MSB1 interacting 1 (BEM1p) during the regulation of the yeast budding process via its OPR/PC/AID (OPCA) motif. Here, we present the structure of an N-terminally truncated version of the Sc CDC24p PB1 domain. It shows a different topology of the beta-sheet than the long form. However, the C-terminal part of the structure shows the conserved PB1 domain features including the OPCA motif with a slight rearrangement of helix alpha1. Residues which are important for the heterodimerization with BEM1p are structurally preserved.     

Membrane-Induced Structure of Scyliorhinin I: A Dual NK1/NK2 Agonist

Scyliorhinin I, a linear decapeptide, is the only known tachykinin that shows high affinity for both NK-1 and NK-2 binding sites and low affinity for NK-3 binding sites. As a first step to understand the structure-activity relationship, we report the membrane-induced structure of scyliorhinin I with the aid of circular dichroism and 2D-(1)H NMR spectroscopy. Sequence specific resonance assignments of protons have been made from correlation spectroscopy (TOCSY, DQF-COSY) and NOESY spectroscopy. The interproton distance constraints and dihedral angle constraints have been utilized to generate a family of structures using DYANA. The superimposition of 20 final structures has been reported with backbone pairwise root mean-square deviation of 0.38 +/- 0.19 A. The results show that scyliorhinin I exists in a random coil state in aqueous environments, whereas helical conformation is induced toward the C-terminal region of the peptide (D4-M10) in the presence of dodecyl phosphocholine micelles. Analysis of NMR data is suggestive of the presence of a 3(10)-helix that is in equilibrium with an alpha-helix in this region from residue 4 to 10. An extended highly flexible N-terminus of scyliorhinin I displays some degree of order and a possible turn structure. Observed conformational features have been compared with respect to that of substance P and neurokinin A, which are endogenous agonists of NK-1 and NK-2 receptors, respectively.     

Structural features of peptoid–peptide hybrids in lipid–water interfaces

The inclusion of peptoid monomers into antimicrobial peptides (AMPs) increases their proteolytic resistance, but introduces conformational flexibility (reduced hydrogen bonding ability and cis/trans isomerism). We here use NMR spectroscopy to answer how the insertion of a peptoid monomer influences the structure of a regular α-helical AMP upon interaction with a dodecyl phosphocholine (DPC) micelle. Insertion of [(2-methylpropyl)amino]acetic acid in maculatin-G15 shows that the structural change and conformational flexibility depends on the site of insertion. This is governed by the micelle interaction of the amphipathic helices flanking the peptoid monomer and the side chain properties of the peptoid and its preceding residue.     

Effects of phenylalanine substitutions in gramicidin A on the kinetics of channel formation in vesicles and channel structure in SDS micelles

The common occurrence of Trp residues at the aqueous-lipid interface region of transmembrane channels is thought to be indicative of its importance for insertion and stabilization of the channel in membranes. To further investigate the effects of Trp-->Phe substitution on the structure and function of the gramicidin channel, four analogs of gramicidin A have been synthesized in which the tryptophan residues at positions 9, 11, 13, and 15 are sequentially replaced with phenylalanine. The three-dimensional structure of each viable analog has been determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. These phenylalanine analogs adopt a homodimer motif, consisting of two beta6.3 helices joined by six hydrogen bonds at their NH2-termini. The replacement of the tryptophan residues does not have a significant effect on the backbone structure of the channels when compared to native gramicidin A, and only small effects are seen on side-chain conformations. Single-channel conductance measurements have shown that the conductance and lifetime of the channels are significantly affected by the replacement of the tryptophan residues (Wallace, 2000; Becker et al., 1991). The variation in conductance appears to be caused by the sequential removal of a tryptophan dipole, thereby removing the ion-dipole interaction at the channel entrance and at the ion binding site. Channel lifetime variations appear to be related to changing side chain-lipid interactions. This is supported by data relating to transport and incorporation kinetics.     

Probing the structure of the Ff bacteriophage major coat protein transmembrane helix dimer by solution NMR

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively ¹⁵N-labeled peptide containing the TM segment of MCP (MCP_TM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCP_TM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.     

Domain Features of the Peripheral Stalk Subunit H of the Methanogenic A1AO ATP Synthase and the NMR Solution Structure of H1-47

A series of truncated forms of subunit H were generated to establish the domain features of that protein. Circular dichroism analysis demonstrated that H is divided at least into a C-terminal coiled-coil domain within residues 54-104, and an N-terminal domain formed by adjacent α-helices. With a cysteine at the C-terminus of each of the truncated proteins (H_1-47, H_1-54, H_1-59, H_1-61, H_1-67, H_1-69, H_1-71, H_1-78, H_1-80, H_1-91, and H_47-105), the residues involved in formation of the coiled-coil interface were determined. Proteins H_1-54, H_1-61, H_1-69, and H_1-80 showed strong cross-link formation, which was weaker in H_1-47, H_1-59, H_1-71, and H_1-91. A shift in disulfide formation between cysteins at positions 71 and 80 reflected an interruption in the periodicity of hydrophobic residues in the region ₇₁AEKILEETEKE₈₁. To understand how the N-terminal domain of H is formed, we determined for the first time, to our knowledge, the solution NMR structure of H_1-47, which revealed an α-helix between residues 15-42 and a flexible N-terminal stretch. The α-helix includes a kink that would bring the two helices of the C-terminus into the coiled-coil arrangement. H_1-47 revealed a strip of alanines involved in dimerization, which were tested by exchange to single cysteines in subunit H mutants.     

The interactions of antimicrobial peptides derived from lysozyme with model membrane systems

Two peptides, RAWVAWR-NH₂ and IVSDGNGMNAWVAWR-NH₂, derived from human and chicken lysozyme, respectively, exhibit antimicrobial activity. A comparison between the L-RAWVAWR, D-RAWVAWR, and the longer peptide has been carried out in membrane mimetic conditions to better understand how their interaction with lipid and detergent systems relates to the reported higher activity for the all L-peptide. Using CD and 2D ¹H NMR spectroscopy, the structures were studied with DPC and SDS micelles. Fluorescence spectroscopy was used to study peptide interactions with POPC and POPG vesicles and DOPC, DOPE, and DOPG mixed vesicle systems. Membrane-peptide interactions were also probed by ITC and DSC. The ability of fluorescein-labeled RAWVAWR to rapidly enter both E. coli and Staphylococcus aureus was visualized using confocal microscopy. Reflecting the bactericidal activity, the long peptide interacted very weakly with the lipids. The RAWVAWR-NH₂ peptides preferred lipids with negatively charged headgroups and interacted predominantly in the solvent-lipid interface, causing significant perturbation of membrane mimetics containing PG headgroups. Peptide structures determined by ¹H NMR indicated a well-ordered coiled structure for the short peptides and the C-terminus of the longer peptide. Using each technique, the two enantiomers of RAWVAWR-NH₂ interacted in an identical fashion with the lipids, indicating that any difference in activity in vivo is limited to interactions not involving the membrane lipids.     

Structural flexibility and the positive charges are the key factors in bacterial cell selectivity and membrane penetration of peptoid-substituted analog of Piscidin 1

Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic α-helical structure isolated from the mast cells of hybrid striped bass. In our previous study, we showed that Pis-1[PG] with a substitution of Pro⁸ for Gly⁸ in Pis-1 had higher bacterial cell selectivity than Pis-1. We designed peptoid residue-substituted peptide, Pis-1[NkG], in which Gly⁸ of Pis-1 was replaced with Nlys (Lys peptoid residue). Pis-1[NkG] had higher antibacterial activity and lower cytotoxicity against mammalian cells than Pis-1 and Pis-1[PG]. We determined the tertiary structure of Pis-1[PG] and Pis-1[NkG] in the presence of DPC micelles by NMR spectroscopy. Both peptides had a three-turn helix in the C-terminal region and a bent structure in the center. Pis-1[PG] has a rigid bent structure at Pro⁸ whereas Pis-1[NkG] existed as a dynamic equilibrium of two conformers with a flexible hinge structure at Nlys⁸. Depolarization of the membrane potential of Staphylococcus aureus and confocal laser-scanning microscopy study revealed that Pis-1[NkG] effectively penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas Pis-1[PG] did not penetrate the membrane but remained outside or on the cell surface. Introduction of a lysine peptoid at position 8 of Pis-1 provided conformational flexibility and increased the positive charge at the hinge region; both factors facilitated penetration of the bacterial cell membrane and conferred bacterial cell selectivity on Pis-1[NkG].