Comparison of interactions between beta-hairpin decapeptides and SDS/DPC micelles from experimental and simulation data

<p>Abstract</p> <p>Background</p> <p>We applied a combined experimental and computational approach to ascertain how peptides interact with host and microbial membrane surrogates, in order to validate simulation methodology we hope will enable the development of insights applicable to the design of novel antimicrobial peptides. We studied the interactions of two truncated versions of the potent, but cytotoxic, antimicrobial octadecapeptide protegrin-1, PC-72 [LCYCRRRFCVC] and PC-73 [CYCRRRFCVC].</p> <p>Results</p> <p>We used a combination of FTIR, fluorescence spectroscopy and molecular dynamics simulations to examine the peptides' interactions with sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles. The relative amounts of secondary structure determined by FTIR agreed with those from the simulations. Fluorescence spectroscopy, deuterium exchange experiments and the simulations all indicate that neither peptide embeds itself deeply into the micelle core. Although molecular simulations placed both peptides at the micelle-water interface, further examination revealed differences in how certain residues interacted with the micelle core.</p> <p>Conclusion</p> <p>We demonstrate here the accuracy of molecular dynamics simulations methods through comparison with experiments, and have used the simulation results to enhance the understanding of how these two peptides interact with the two types of micelles. We find agreement between simulation and experimental results in the final structure of the peptides and in the peptides final conformation with respect to the micelle. Looking in depth at the peptide interactions, we find differences in the interactions between the two peptides from the simulation data; Leu-1 on PC-72 interacts strongly with the SDS micelle, though the interaction is not persistent – the residue withdraws and inserts into the micelle throughout the simulation.</p

Tug of War in Lung Surfactant Components: MiniB Dominates over Cholesterol during Lipid Domain Formation

This is the published version. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.Lung surfactants (LS), a complex mixture of lipids and proteins present in the alveolar lining of lungs, help in lowering surface tension to near zero at expiration. Deficiency of this surfactant can lead to Neonatal Respiratory Distress Syndrome in infants, while a dysfunction of LS can cause Acute Respiratory Distress Syndrome (ARDS) that affects patients irrespective of age. Successful medical intervention such as surfactant replacement therapy (SRT) requires a good understanding of surfactant composition and function. Currently there is no consensus on the composition of LS used in SRT, particularly the interactions between components making up this mixture. Our objective was to understand the interaction of cholesterol (a component whose role and even presence in SRT is highly debated) and MiniB (a synthetic protein mimic of native surfactant protein SP-B) at air-water interface. We report the alteration in lipid domain formation of films containing 1,2-dipalmitoyl- sn- glycero- 3- phosphocholine (DPPC): 1- palmitoyl- 2- oleoyl- sn- glycero- 3- phosphatidylglycerol (POPG) in the ratio 7:3 under the influence of varying concentrations of MiniB and cholesterol. Fluorescence imaging under constant compression, along with analysis of domain size distributions, reveals that MiniB increases line tension between lipid domains, and prefers to stay in fluid POPG regions, making the liquid-ordered domains smaller in size. Small amounts of cholesterol prefer packed domains, stretching them into spirals during the process, lowering their line tension. In both cases, higher concentration yields more prominent consequences in terms of the stated changes. However, mixture containing both cholesterol and MiniB shows reduction in domain size with no changes in domain shape. This suggests the dominance of MiniB over cholesterol when interacting with lipid domains, which may have important effects on the performance of synthetic LS

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1–25 and SP-B8–25

AbstractRaman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B1–25). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B1–25 was compared with that of a truncated homodimer (dSP-B8–25) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S–S stretching region centered at ∼500 cm−1 indicated a stable, although highly strained disulfide conformation with a χ(CS–SC) dihedral angle of ±10° for the dSP-B1–25 dimer. In contrast, the truncated dimer dSP-B8–25 exhibited a series of disulfide conformations with the χ(CS–SC) dihedral angle taking on values of either ±30° or 85±20°. For conformations with χ(CS–SC) close to the ±90° value, the Raman spectra of the 8–25 truncated dimers exhibited χ(SS–CC) dihedral angles of 90/180° and 20–30°. In the presence of a lipid mixture, both constructs showed a ν(S–S) band at ∼488 cm−1, corresponding to a χ(CS–SC) dihedral angle of ±10°. Polarized infrared spectroscopy was also used to determine the orientation of the helix and β-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of ∼60–70° to the surface normal, while the β structure had ∼40° tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation

Membrane interactions of the synthetic N-terminal peptide of HIV-1 gp41 and its structural analogs

AbstractStructural and functional studies assessed the membrane actions of the N terminus of HIV-1 glycoprotein 41 000 (gp41). Earlier site-directed mutagenesis has shown that key amino acid changes in this gp41 domain inhibit viral infection and syncytia formation. Here, a synthetic peptide corresponding to the N terminus of gp41 (FP; 23 residues, 519–541), and also FP analogs (FP520V/E with Val→Glu at residue 520; FP527L/R with Leu→Arg at 527; FP529F/Y with Phe→Tyr at 529; and FPCLP1 with FP truncated at 525) incorporating these modifications were prepared. When added to human erythrocytes at physiologic pH, the lytic and aggregating activities of the FP analogs were much reduced over those with the wild-type FP. With resealed human erythrocyte ghosts, the lipid-mixing activities of the FP analogs were also substantially depressed over that with the wild-type FP. Combined with results from earlier studies, theoretical calculations using hydrophobic moment plot analysis and physical experiments using circular dichroism and Fourier transform infrared spectroscopy indicate that the diminished lysis and fusion noted for FP analogs may be due to altered peptide-membrane lipid interactions. These data confirm that the N-terminal gp41 domain plays critical roles in the cytolysis and fusion underlying HIV-cell infection

Conformation and Trimer Association of the Transmembrane Domain of the Parainfluenza Virus Fusion Protein in Lipid Bilayers from Solid-State NMR: Insights into the Sequence Determinants of Trimer Structure and Fusion Activity

Enveloped viruses enter cells by using their fusion proteins to merge the virus lipid envelope and the cell membrane. While crystal structures of the water-soluble ectodomains of many viral fusion proteins have been determined, the structure and assembly of the C-terminal transmembrane domain (TMD) remains poorly understood. Here we use solid-state NMR to determine the backbone conformation and oligomeric structure of the TMD of the parainfluenza virus 5 fusion protein. 13C chemical shifts indicate that the central leucine-rich segment of the TMD is α-helical in POPC/cholesterol membranes and POPE membranes, while the Ile- and Val-rich termini shift to the β-strand conformation in the POPE membrane. Importantly, lipid mixing assays indicate that the TMD is more fusogenic in the POPE membrane than in the POPC/cholesterol membrane, indicating that the β-strand conformation is important for fusion by inducing membrane curvature. Incorporation of para-fluorinated Phe at three positions of the α-helical core allowed us to measure interhelical distances using 19F spin diffusion NMR. The data indicate that, at peptide:lipid molar ratios of ~ 1:15, the TMD forms a trimeric helical bundle with inter-helical distances of 8.2–8.4 Å for L493F and L504F and 10.5 Å for L500F. These data provide high-resolution evidence of trimer formation of a viral fusion protein TMD in phospholipid bilayers, and indicate that the parainfluenza virus 5 fusion protein TMD harbors two functions: the central α-helical core is the trimerization unit of the protein, while the two termini are responsible for inducing membrane curvature by transitioning to a β-sheet conformation. Keywords: magic-angle-spinning NMR; trimer formation; conformational plasticity; spin diffusionNational Institutes of Health (U.S.) (Grant GM066976

A sulfur-free peptide mimic of surfactant protein B (B-YL) exhibits high in vitro and in vivo surface activities [version 2; referees: 2 approved]

Background: Animal-derived surfactants containing surfactant proteins B (SP-B) and C (SP-C) are used to treat respiratory distress syndrome (RDS) in preterm infants. SP-B (79 residues) plays a pivotal role in lung function and the design of synthetic lung surfactant. Super Mini-B (SMB), a 41-residue peptide based on the N- and C-domains of SP-B covalently joined with a turn and two disulfides, folds as an α-helix hairpin mimicking the properties of these domains in SP-B. Here, we studied ‘B-YL’, a 41-residue SMB variant that has its four cysteine and two methionine residues replaced by tyrosine and leucine, respectively, to test whether these hydrophobic substitutions produce a surface-active, α-helix hairpin. Methods: Structure and function of B-YL and SMB in surfactant lipids were compared with CD and FTIR spectroscopy, and surface activity with captive bubble surfactometry and in lavaged, surfactant-deficient adult rabbits. Results: CD and FTIR spectroscopy of B-YL in surfactant lipids showed secondary structures compatible with peptide folding as an α-helix hairpin, similar to SMB in lipids. B-YL in surfactant lipids demonstrated excellent in vitro surface activity and good oxygenation and dynamic compliance in lavaged, surfactant-deficient adult rabbits, suggesting that the four tyrosine substitutions are an effective replacement for the disulfide-reinforced helix-turn of SMB. Here, the B-YL fold may be stabilized by a core of clustered tyrosines linking the N- and C-helices through non-covalent interactions involving aromatic rings. Conclusions: ‘Sulfur-free’ B-YL forms an amphipathic helix-hairpin in surfactant liposomes with high surface activity and is functionally similar to SMB and native SP-B. The removal of the cysteines makes B-YL more feasible to scale up production for clinical application. B-YL’s possible resistance against free oxygen radical damage to methionines by substitutions with leucine provides an extra edge over SMB in the treatment of respiratory failure in preterm infants with RDS

Surfactant protein C peptides with salt-bridges (“ion-locks”) promote high surfactant activities by mimicking the α-helix and membrane topography of the native protein

Background. Surfactant protein C (SP-C; 35 residues) in lungs has a cationic N-terminal domain with two cysteines covalently linked to palmitoyls and a C-terminal region enriched in Val, Leu and Ile. Native SP-C shows high surface activity, due to SP-C inserting in the bilayer with its cationic N-terminus binding to the polar headgroup and its hydrophobic C-terminus embedded as a tilted, transmembrane α-helix. The palmitoylcysteines in SP-C act as ‘helical adjuvants’ to maintain activity by overriding the β-sheet propensities of the native sequences.Objective. We studied SP-C peptides lacking palmitoyls, but containing glutamate and lysine at 4-residue intervals, to assess whether SP-C peptides with salt-bridges (“ion-locks”) promote surface activity by mimicking the α-helix and membrane topography of native SP-C.Methods. SP-C mimics were synthesized that reproduce native sequences, but without palmitoyls (i.e., SP-Css or SP-Cff, with serines or phenylalanines replacing the two cysteines). Ion-lock SP-C molecules were prepared by incorporating single or double Glu−–Lys+ into the parent SP-C’s. The secondary structures of SP-C mimics were studied with Fourier transform infrared (FTIR) spectroscopy and PASTA, an algorithm that predicts β-sheet propensities based on the energies of the various β-sheet pairings. The membrane topography of SP-C mimics was investigated with orientated and hydrogen/deuterium (H/D) exchange FTIR, and also Membrane Protein Explorer (MPEx) hydropathy analysis. In vitro surface activity was determined using adsorption surface pressure isotherms and captive bubble surfactometry, and in vivo surface activity from lung function measures in a rabbit model of surfactant deficiency.Results. PASTA calculations predicted that the SP-Css and SP-Cff peptides should each form parallel β-sheet aggregates, with FTIR spectroscopy confirming high parallel β-sheet with ‘amyloid-like’ properties. The enhanced β-sheet properties for SP-Css and SP-Cff are likely responsible for their low surfactant activities in the in vitro and in vivo assays. Although standard 12C-FTIR study showed that the α-helicity of these SP-C sequences in lipids was uniformly increased with Glu−–Lys+ insertions, elevated surfactant activity was only selectively observed. Additional results from oriented and H/D exchange FTIR experiments indicated that the high surfactant activities depend on the SP-C ion-locks recapitulating both the α-helicity and the membrane topography of native SP-C. SP-Css ion-lock 1, an SP-Css with a salt-bridge for a Glu−–Lys+ ion-pair predicted from MPEx hydropathy calculations, demonstrated enhanced surfactant activity and a transmembrane helix simulating those of native SP-C.Conclusion. Highly active SP-C mimics were developed that replace the palmitoyls of SP-C with intrapeptide salt-bridges and represent a new class of synthetic surfactants with therapeutic interest

Calcitermin, a novel antimicrobial peptide isolated from human airway secretions

AbstractThe human airways are protected from pathogenic colonization by a blanket of fluid impregnated with innate antimicrobial effector molecules. Among several previously uncharacterized components, we isolated a peptide that had activity primarily targeting Gram-negative bacteria. We named the peptide ‘calcitermin’ since its amino acid sequence and mass were equivalent to the 15 C-terminal residues of the S100 protein, calgranulin C. The antimicrobial activity of calcitermin was enhanced in acidic buffers (pH 5.4) and in the presence of micromolar concentrations of ZnCl2. Analysis revealed a putative zinc-binding consensus sequence as well as an α-helical conformation in structure-promoting solvents

Structural correlates of antimicrobial efficacy in IL-8 and related human kinocidins

AbstractChemokines are small (8–12 kDa) effector proteins that potentiate leukocyte chemonavigation. Beyond this role, certain chemokines have direct antimicrobial activity against human pathogenic organisms; such molecules are termed kinocidins. The current investigation was designed to explore the structure–activity basis for direct microbicidal activity of kinocidins. Amino acid sequence and 3-dimensional analyses demonstrated these molecules to contain iterations of the conserved γ-core motif found in broad classes of classical antimicrobial peptides. Representative CXC, CC and C cysteine-motif-group kinocidins were tested for antimicrobial activity versus human pathogenic bacteria and fungi. Results demonstrate that these molecules exert direct antimicrobial activity in vitro, including antibacterial activity of native IL-8 and MCP-1, and microbicidal activity of native IL-8. To define molecular determinants governing its antimicrobial activities, the IL-8 γ-core (IL-8γ) and α-helical (IL-8α) motifs were compared to native IL-8 for antimicrobial efficacy in vitro. Microbicidal activity recapitulating that of native IL-8 localized to the autonomous IL-8α motif in vitro, and demonstrated durable microbicidal activity in human blood and blood matrices ex vivo. These results offer new insights into the modular architecture, context-related deployment and function, and evolution of host defense molecules containing γ-core motifs and microbicidal helices associated with antimicrobial activity