Structure of Transmembrane Pores Stabilized by Antimicrobial Peptides Magainin and PGLa

Wednesday, February 11, 2015 2782-Pos Board B212 Molecular Dynamics Simulations Reveal Mechanistic Details of Polymyxin Penetration into both Membranes of E. coli Syma Khalid1, Nils A. Berglund1, Peter J. Bond2, Thomas J. Piggot3. 1 School of Chemistry, University of Southampton, UK, Southampton, United Kingdom, 24 Bioinformatics Institute (A*STAR), Singapore, Singapore, 3 Detection Department, Defence Science and Technology Laboratory, Salisbury, United Kingdom. Gram-negative bacteria such as E.coli are protected by a surprisingly complex cell envelope. The cell envelope is composed of two membranes that form a protective barrier around the cells, and control the influx and efflux of solutes via various routes. Lysing the cell by disrupting the membranes, or permeating across them to gain access to the cell interior are key properties for antimicrobial agents. Polymyxins are a class of antibiotics that have been shown to be highly active against Gram-negative bacteria. It is thought they enter bacterial cells through a self-uptake mechanism, although the molecular details of the mechanism are still unclear. We present, to our knowledge, the first molecular dynamics simulation study of an antimicrobial peptide, with both membranes of E.coli. Our model of the outer membrane contains lipopolysaccharide molecules in the inner leaflet and a mixture of ohosphilipids in the inner leaflet. In contrast, the inner membrane is comprised only of phospholipids. Our simulations reveal the effects of Polymyxin B1 (PMB1) binding on the physical properties of each membrane. Thus they are able to identify potentially different mechanisms for membrane disruption by PMB1. Peptide aggregation and insertion of one peptide tail was observed in the outer membrane. In contrast, PMB1 peptides insert readily as monomers, accompanied by water penetration into the inner membrane. Our simulations demonstrate the importance of capturing relevant details of biological complexity, in molecular models of biological membrane systems. 2783-Pos Board B213 Thermodynamics Govern the Mechanism of Antimicrobial Lipopeptides: Insights from Coarse-Grained Molecular Dynamics Simulations Dejun Lin, Alan Grossfield. University of Rochester, Rochester, NY, USA. Antimicrobial lipopeptides (AMLPs) are a series of acylated cationic peptides with broad-spectrum antimicrobial activity and low hemolytic activity. We used microsecond-scale coarse-grained molecular dynamics simulations with the MARTINI force field to understand AMLPs’ modes of action. Using rigorous free energy calculations with a novel reaction coordinate, we quantified formation of lipopeptide micelles in solution, as well as the the affinity of those micelles for different membrane compositions. The results yield both kinetic and thermodynamics arguments for the lipopeptides selectivity for bacterial over mammalian membranes. Our results indicate that the acyl chain of C16-KGGK, one of the AMLPs, is mainly responsible for its affinity to membrane while the peptide portion determines the selectivity towards different membrane lipid compositions. The micelle results suggest both kinetic and thermodynamics arguments for the lipopeptides selectivity for bacterial over mammalian membranes. Our results provide biophysical insights into the mechanism of lipopeptides’ antimicrobial action. 2784-Pos Board B214 The Nature of Daptomycin Aggregates Ming-Tao Lee1,2, Wei-Chin Hung3, Yen-Fei Chen4, Huey W. Huang4. 1 Life Science Group, National Synchrotron Radiation Reasearch Center, Hsinchu, Taiwan, 2Department of Physics, National Central University, Jhongli, Taiwan, 3Department of Physics, R. O. C. Military Academy, Fengshan, Taiwan, 4Department of Physics & Astronomy, Rice University, Houston, TX, USA. Daptomycin is the first FDA-approved member of a new structural class of antibiotics_the cyclic lipopeptides. It is an important drug against multidrugresistant gram-positive pathogens. The peptide interacts with the lipid matrix of cell membranes, inducing membrane permeability to ions, but its molecular mechanism has been a puzzle. Unlike the ubiquitous membrane-acting host-defense antimicrobial peptides, daptomycin does not induce molecular-leaking pores in the cell membranes–no calcein leakage was detected from lipid vesicles. Thus how it affects the membrane permeability to ions is not clear. The antibacterial activity and induced ion leakage by daptomycin correlate with the target membrane’s content of phosphatidylglycerol (PG) and occur only in the presence of Ca2þ ions. Fluorescence resonance energy transfer (FRET) experiments and the recently discovered lipid extracting effect have shown daptomycin aggregation in membranes, but the chemical structure of daptomycin gives no clue to the nature of daptomycin aggregates. Jung et al discovered an inversion of the CD spectrum of daptomycin that occurs only in the simultaneous presence of PG and Caþþ. We have correlated the inversion of the CD

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with the lipid extracting effect of daptomycin. Here we make use of the CD spectral change with Ca2þ ion concentration and with PG concentration to analyze the nature of the daptomycin-Caþþ-PG aggregates. The data shows a sharpe threshold (or micellization) effect on the Ca2þ ion concentration dependence, indicating that each aggregate contains 20 or more calcium ions. This result shows that the aggregates are large, inconsistent with the idea of daptomycin aggregates forming oligomeric pores, since such a pore must be made of a relatively small number of daptomycin. Once we realize the micellization effect, the stoichiometry of the daptomycin-Caþþ-PG aggregates can be analyzed from the calcium and PG concentration dependence. 2785-Pos Board B215 Lipid Selectivity of Fungicidal Lipopeptides Sebastian Fiedler, Quang Huynh, Hiren Patel, Heiko Heerklotz. Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada. Cyclic lipopeptides act against a variety of pathogens and, thus, constitute highly efficient crop-protection agents. For example, commercially available fungicides contain mixtures of different Bacillus subtilis lipopeptide families, such as surfactins (SF), iturins (IT), and fengycins (FE). Besides other effects, the fungicidal activity of these peptides is mainly mediated by permeabilizing the membrane. Accordingly, surfactins behave like extremely powerful surfactants and induce graded membrane leakage, iturins synergize with surfactins, and fengycins follow an all-or-none leakage mechanism. However, little is known about how the lipid composition of the membrane affects the capability of these lipopeptides to induce membrane leakage. To shed more light on their lipid selectivity, we performed fluorescence-lifetime-based leakage assays on various types of lipidbilayer compositions. To this end, we compare leakage efficiencies and mode of actions of SF, IT, FE, and mixtures of them in lipid vesicles composed of phosphatidylcholine/phosphatidylethanolamine (PC/PE), PE/phosphatidylglycerol (PE/PG), and analyze the effect of phosphatidylserine and cardiolipin. Furthermore, we assessed the influence of ergosterol as a main component of fungal cell membranes. Our results aid in the understanding of the mechanism of lipopeptide-induced fungicidal activity and demonstrate that B. subtilis tailors these lipopeptide mixtures to specifically attack fungal membranes. 2786-Pos Board B216 Structure and Membrane Topology of the Pore-Forming Peptide Maculatin 1.1 Marc-Antoine Sani1, Terry P. Lybrand2, Frances Separovic1. 1 School of Chemistry Bio21 institute, Univeristy of Melbourne, parkville, Australia, 2Center for Structural Biology, Vanderbilt University, Nashville, TN, USA. Antimicrobial peptides (AMP) that target membranes are an attractive alternative to classic antibiotics, since they do not require internalization nor target a specific stereo-structure, thus limiting development of bacterial resistance. Their mode of action involves the disruption of lipid membranes; however, the relationship between lipid membrane structure and peptide potency remains unclear. We present the structural investigation of the AMP maculatin 1.1 (Mac1) in DPC micelles and DHPC/DMPC isotropic (q¼ 0.5) bicelles. Using solution and solidstate NMR with paramagnetic relaxation enhancement agents (PRE) and molecular dynamics (MD), we demonstrate the important role of the membrane structure in modulating the structure and location of Mac1. HSQC of specifically 15N labeled Mac1 in buffer displayed a narrow chemical shift dispersion that is typical of random coil structures. Introduction of micelles and bicelles produced chemical shift dispersions characteristic of helical structures, with differences suggesting that Mac1 adopts a different degree of helicity dependent on the curvature. 3D TROSY-NOESY allowed assignment of the sequential 15N labeled residues, and determination of a 3D helical structure in phospholipid micelles and bicelles, the latter producing the greatest helical stretch. Titration of the PRE agent Gd3þ-(DTPA) showed that the central core of Mac1 is protected in bicelles while in micelles only the N-term is exposed to the PRE effect. MD simulations in DPC micelles revealed N-term exposure to the solvent, and they also suggested that Mac1 bent to adapt the curved micelle structure. Experiments will be repeated with 4 and 8 peptides per micelles. 2787-Pos Board B217 Structure of Transmembrane Pores Stabilized by Antimicrobial Peptides Magainin and PGLa Almudena Pino Angeles, John M. Leveritt III,, Themis Lazaridis. Chemistry, City College of New York, New York, NY, USA. Antimicrobial peptides are found in many organisms as part of their defense system against bacterial infections. They have similar structural and functional features: most consist of amphiphilic helices that bind to a membrane and disrupt it by diverse methods. Among these peptides, magainin 2 and PGLa are found in the frog skin and exhibit synergistic effects in lipid bilayer disruption by the

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Wednesday, February 11, 2015

formation of transmembrane pores [1]. Experimental methods, such as solidstate NMR, have shed light on the relative orientation of magainin 2 and PGLa on membranes with different lipid composition [2,3], but there is a general lack of information on the structure of the pore and the specific interactions that lead to its stabilization. In the present work, we studied the structure and dynamics of transmembrane pores formed by magainin 2 and magainin 2/PGLa (2:2) tetramers by all-atom molecular dynamics simulations performed at the Anton supercomputer (Pittsburgh Supercomputing Center). For both systems we observed stabilization of a pore. The 9-ms simulation time allows a detailed analysis of its structure and properties, the role of the lipids surrounding it, and the relative orientation of magainin 2 and PGLa in the membrane. References [1] Matsuzaki, Mikani, Akada et al. Biochemistry (1998) 37:15144-15153 [2] Tremouilhac P, Strandberg E, Wadhwani P and Ulrich AS. J. Biol. Chem. (2006) 281:32089-32094 [3] Strandberg E, Zerweck J, Wadhwani P and Ulrich AS. Biophys. J. (2013) 104: L09-L011 2788-Pos Board B218 Modulation of the Interaction Between Detergent Micelles and Model Peptide Antibiotics by Varying the Peptide Charge Distribution John Weirich1, Brianna Haight1, Olivier Lequin2, Lucie Khemte´mourian2, Ludovic Carlier2, Adrienne Loh1. 1 Chemistry & Biochemistry, U. Wisconsin - La Crosse, La Crosse, WI, USA, 2 Laboratoire des Biomole´cules, UMR 7203 UPMC-ENS-CNRS, Universite´ Pierre et Marie Curie, Paris, France. With rising disease rates and decreasing effectiveness of conventional antibiotics, there is an immediate need for new antibiotics. One promising solution is through cationic antimicrobial peptides, which act by perturbing bacterial membranes. We are investigating model peptide antibiotics composed primarily of the hydrophobic dialkylated amino acid Aib (a-aminoisobutyric acid), which imparts a strong 310-helical bias due to steric hindrance at the a-carbon. Cationic lysine residues were placed in adjacent locations in the center of the helix (KK45) or one full turn apart (KK36). Micelles of dodecylphosphocholine (DPC) or sodium dodecyl sulfate (SDS) were used as zwitterionic or anionic membrane models, respectively. The interaction of model peptides with micelles can provide valuable information about the role of helical structure and peptide charge distribution on peptide-membrane interactions. Here we present thermodynamic and spectroscopic data characterizing the peptide-micelle interactions. Binding enthalpies for the interactions of KK36 and KK45 with DPC and SDS micelles were measured using isothermal titration calorimetry (ITC). Preliminary data suggests that binding to SDS micelles is exothermic, while binding to DPC micelles is endothermic. In both cases, KK45 has a more favorable binding enthalpy than KK36. Measurements of longitudinal relaxation times (T1) in the absence and presence of a gadolinium line broadening reagent indicate that KK45 is more buried than KK36 in SDS micelles, and that both peptides are more buried in SDS micelles than in DPC micelles. These results suggest that the enthalpy of binding is dominated by hydrophobic interactions between the Aib sidechains and the detergent molecules. These interactions are enhanced in KK45, possibly because the charges are more localized to the center of the helix. 2789-Pos Board B219 Isomeric Model Antibiotic Peptides Differing Only in Charge Placement Adopt Different Helical Conformations Jayna Sharma1, Riley Larson1, Olivier Lequin2, Lucie Khemte´mourian2, Ludovic Carlier2, Kevin Larsen3, Theodore Savage4, Adrienne Loh1. 1 Chemistry & Biochemistry, U. Wisconsin - La Crosse, La Crosse, WI, USA, 2 Laboratoire des Biomole´cules, UMR 7203 UPMC-ENS-CNRS, Universite´ Pierre et Marie Curie, Paris, France, 3Biophysics Program, Stanford University School of Medicine, Stanford, CA, USA, 4Wisconsin State Lab of Hygiene, Madison, WI, USA. The efficacy of existing antibiotics is slowly declining as species of bacteria are evolving, increasing the need to find new antibiotics. One promising opportunity lies in peptide antibiotics, which are commonly helical and cationic. Our model antibiotic peptides are composed primarily of the hydrophobic dialkylated amino acid Aib (a -aminoisobutyric acid), which imparts a strong 310-helical bias due to steric hindrance at the a-carbon. Cationic lysine residues are substituted into strategic locations in the sequence. Previous studies have reported that substitution of monoalkylated amino acids into an Aib-rich sequence can impact helical shape. We report here the effect of charge placement on peptide helical structure in two solvents: DMSO (dimethyl sulfoxide), and water. We are focusing on two octameric peptides, in which two lysine residues are placed in adjacent locations in the sequence (KK45), or one turn apart (KK36). NMR data indicates that in DMSO, KK36 adopts a canonical 310-helical structure, while the KK45 helix is kinked. However, circular dichroism

spectra and NMR measurements of amide temperature coefficients for aqueous KK36 are not consistent with a canonical 310-helical conformation. We present here the complete titration of amide proton chemical shifts for KK36 and KK45 from aqueous (90:10 H2O/D2O) solution to DMSO-d6 in order to identify the residues that are undergoing local environment changes as the bulk environment is changed. Measurements of the CD spectra of both peptides aqueous solution as a function of temperature will be correlated with the amide temperature coefficient data from NMR to obtain a more complete picture of solventinduced conformational changes. Ultimately the correlation of structure and sequence will inform future antibiotic peptide design. 2790-Pos Board B220 Anisotropic Membrane Curvature Sensing by Antibacterial Peptides Jordi Go´mez-Llobregat1, Martin Linde´n2. 1 Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden, 2 Cell and Molecular Biology, Uppsala University, Uppsala, Sweden. Some proteins and peptides have an intrinsic capacity to remodel lipid bilayers and sense membrane curvature via a curvature-dependent membrane binding energy. This is crucial for many biological processes. For example, antimicrobial peptides are believed to disrupt bacterial membranes by producing pores, which are highly curved structures. In this work, we explore a new computational method to investigate curvature sensing by simulating the interaction of single peptides with a buckled lipid bilayer, using the coarse-grained Martini model. We analyze three canonical antimicrobial peptides, magainin, melittin, and LL37, and find qualitatively different sensing characteristics. In particular, melittin and LL-37 show anisotropic curvature sensitivity, but with different preferred orientations relative to the direction of greatest curvature. These findings provides new insights into the microscopic mechanisms of curvature sensing and its role in membrane remodeling, and should motivate experimental development to simultaneously measure position and orientation of membrane-bound proteins. 2791-Pos Board B221 Membrane Interaction of an Anti-Bacterial AApeptide Defined by EPR Spectroscopy Pavanjeet Kaur1, Yaqiong Li2, Jianfeng Cai2, Likai Song1. 1 National High Magnetic Field Laboratory and Florida State University, Tallahassee, FL, USA, 2University of South FLorida, Tampa, FL, USA. Antibiotic resistance is one of the major threats to public health. AApeptides are a new class of synthetic anti-bacterial peptidomimetics that are not prone to antibiotic resistance, and are highly resistant to protease degradation. The broadspectrum anti-bacterial activities of AApeptides are believed to be related to their unique structural features, which are capable of disrupting bacterial membranes selectively over human eukaryotic cells. How AApeptides selectively interact with bacterial membranes and alter lipid assembly and properties is unclear, but such information is essential in order to understand their antimicrobial activities. Here, by using electron paramagnetic resonance (EPR) techniques at 9 and 95 GHz, we have characterized the membrane interaction and destabilizing activities of an AApeptide, cyclic-g-AApeptide, on liposomes mimicking bacterial and eukaryotic cell membranes. The analysis revealed specific interactions between cyclic-g-AApeptides and negatively-charged lipid molecules. Subsequently, the AApeptide interacts strongly with the bacteria-mimic liposomes containing negatively-charged lipids, and thereby inhibits membrane fluidity. Furthermore, AApeptide binding induces significant lipid-lateral-ordering of the bacteria-mimic liposomes, detected by EPR at 95 GHz. In addition, AApeptide binding increases the membrane permeability of the bacteriamimic liposomes. By contrast, minimal membrane fluidity and permeability changes were observed for liposomes mimicking eukaryotic cell membranes, consisting of neutral lipids and cholesterol, upon AApeptide binding. The results revealed that the intrinsic features of AApeptides are important for their ability to selectively disrupt bacterial membranes, the implications of which extend to developing new antibacterial biomaterials. 2792-Pos Board B222 Activity of Antimicrobial Peptide Protegrin-1 is Tuned by Membrane Cholesterol Content J. Michael Henderson1, Kathleen D. Cao1, Zhiliang L. Gong1, Gregory T. Tietjen2, Charles T.R. Heffern1, Daniel Kerr3, Nishanth Iyengar1, Indroneil Roy4, Alan J. Waring5,6, Mati Meron7, Binhua Lin7, Sushil Satija8, Jaroslaw Majewski9, Ka Yee C. Lee1. 1 Chemistry, University of Chicago, Chicago, IL, USA, 2School of Engineering & Applied Science, Yale University, New Haven, CT, USA, 3 Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA, 4Chemical Engineering, City College of New York, New York, NY, USA, 5University of California Los Angeles, Los Angeles, CA, USA, 6 University of California Irvine, Irvine, CA, USA, 7Center for Advanced Radiation Sources, University of Chicago, Chicago, IL, USA, 8NIST Center