- Email: [email protected]
An Exact Model of Daptomycin Binding to Lipid Bilayers
24a
Sunday, February 12, 2017
(2) Stranberg E, Zerweck J et al. Biophys J (2013) 104(6):L9-11 (3) Leveritt JM 3rd, Pino-Angeles A and Lazaridis T. Biophys J (2015) 108(10):2424-6 (4) Wiedman G, Fuselier T et al. J Am Chem Soc (2014) 136(12):4724-31 123-Plat Spontaneous Formation of an Ensemble of Structurally Diverse Membrane Channel Architectures from a Single Antimicrobial Peptide Maculatin Yukun Wang1, Charles Chen1, Dan Hu2, Jakob Ulmschneider2, Martin Ulmschneider1. 1 Johns Hopkins University, Baltimore, MD, USA, 2Shanghai Jiaotong University, Shanghai, China. Antimicrobial peptides (AMPs) are an ancient, powerful, and ubiquitous component of the innate immune defence in all domains of life and play a key role in controlling the human microbiome. Many AMPs are known to selectively target and form pores in microbial membranes, killing a wide variety of pathogens at low micromolar concentrations. Fundamental questions remain, however, regarding the molecular mechanisms of membrane targeting, pore formation and function, and the extent to which poration contributes towards antimicrobial activity. Here we report an experimentally guided and validated unbiased long-timescale simulation methodology that yields the complete mechanism of spontaneous pore assembly in the membrane for the amphiphilic pore-forming AMP maculatin at atomic resolution. Rather than a single well defined pore, maculatin was found to form an ensemble of well defined temporarily functional channels that continuously form and dissociate in the membrane. All channels are formed from highly symmetric low-oligomeric assemblies of up to 8 membrane-spanning peptides that mimic integral membrane protein channels in structure. Each channel has a different architecture, functional lifetime, and ion conductivity and overall membrane permeabilization is dominated by higher order oligomers, which form ion channels that also conduct water at high rates. The highest order oligomers were also found to efficiently conduct small dyes. Channel architectures as well as their relative weighting in the ensemble are sensitive to minor mutations as well as variation of lipid tail length. All pores are formed spontaneously by the consecutive addition of individual helices to a transmembrane helix or helix bundle, in contrast to current poration models postulated for AMPs. The diversity of channel architectures formed by a single sequence is remarkable and could explain why no sequence-function relationships in AMP sequences have been discovered to date. Structural ensembles formed by AMP may be the key to preventing bacterial resistance. 124-Plat An Exact Model of Daptomycin Binding to Lipid Bilayers Antje Pokorny, Tala O. Khatib. Chemistry and Biochemistry, Univ. North Carolina Wilmington, Wilmington, NC, USA. Daptomycin is a cyclic lipopeptide of clinical importance in the treatment of multi-drug resistant infections, including those caused by methicillinresistant S. aureus (MRSA) strains. Similar to other antimicrobial peptides, daptomycin binds with preference to the anionic cytoplasmic membranes typically found in prokaryotes. However, in contrast to most linear, alpha-helical peptides, daptomycin binds to lipid bilayers only in the presence of calcium ions and its activity is absolutely calcium-dependent. We measured the interaction of daptomycin with anionic lipid membranes using kinetic binding experiments and equilibrium titrations. The data were analyzed using an exact model describing the interactions of daptomycin with the lipid bilayer that includes solution and membrane-bound states, and the influence of calcium ions on daptomycin-lipid interactions. 125-Plat The Metallopeptides Piscidin 1 and Piscidin 3 Employ Membrane and Nuclease Activity to Eradicate Planktonic, Biofilm, and Persister Cells Myriam L. Cotten1, M. Daben J. Libardo2, Ali Adem Bahar3, Riqiang Fu4, Vitalii I. Silin5, Dacheng Ren3, Mihaela Mihailescu5, Alfredo Angeles-Boza2. 1 Applied Science, College of William and Mary, Williamsburg, VA, USA, 2 Chemistry, University of Connecticut, Storrs, CT, USA, 3Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY, USA, 4National High Magnetic Field Laboratory, Tallahassee, FL, USA, 5Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA. Piscidin 1 (P1) and piscidin 3 (P3) are highly potent host-defense peptides (HDPs) active against drug resistant bacteria, and therefore important for the design of novel anti-infective therapeutics. Both peptides are cationic and interact with the anionic lipid membranes of bacteria. They also translocate across these membranes and colocalize with intracellular DNA. While both peptides have similar three-dimensional structures, P1 is generally more
biologically active than P3 for reasons that are not understood. Here, we compare the disruptive effects of P1 and P3 on lipid bilayers and DNA, and investigate the role of their amino terminal Cu and Ni (ATCUN) binding motif in their mechanism of action. We rely on a combination of complementary methods, including CD- and NMR-monitored titrations, structural solid-state NMR, neutron diffraction with deuterium labeling, surface plasmon resonance, microscopy, biological activity assays, and voltammetry. We find by solid-state NMR that both peptides use an a-helical structure to interact with bacterial cell membrane mimics and isolated DNA. Interestingly, while P1 is more membrane active than P3, the latter is more disruptive to DNA. Through neutron diffraction studies, we demonstrate that P1 inserts more deeply in lipid membranes and induces a major conformational rearrangement of the lipid headgroups, which leads to bilayer defects and significant water penetration into the interstices of the membrane. Remarkably, both peptides bound to Cu2þ act as nucleases that damage isolated DNA within minutes and P3 is significantly more effective than P1. In the context of bacterial cells, we show that copper plays an essential role in planktonic, biofilm, and persister cell eradication. Furthermore, we find that the more membrane-active P1 kills bacteria rapidly while the more DNA-damaging P3 results in slower cell death. Taken together, these results identify host defense metallopeptides with complementary and multi-pronged mechanisms of action as valuable templates to consider for the design of novel strategies against drug resistant bacteria. 126-Plat 20D Years and no End in Sight: Histidine-Rich Designer Peptides with pH-Dependent Membrane Topology and with Multifacet Biomedical Potential Christopher Aisenbrey1, Philippe Bertani1, David Fenard2, Anne Galy3, Elise Glattard1, Martin Gotthardt4, Antoine Kichler5, Nan Liu4, Arnaud Marquette1, Regine S€uss4, Louic Vermeer1, Dennis Wilkins-Juhl1, Justine Wolf1, Burkhard Bechinger1. 1 Chemistry, University of Strasbourg/CNRS, Strasbourg, France, 2Innovation Technologique Lentivirus, Ge´ne´thon, Evry, France, 3Ge´ne´thon/Inserm UMR951, Evry, France, 4Pharmazeutische Technologie und Biopharmazie, University of Freiburg, Freiburg, Germany, 5Laboratoire ‘‘Vecteurs: Synthe`se et Applications The´rapeutiques’’ (V-SAT), CNRS, Strasbourg, France. The synthetic LAH4 peptides were designed to investigate the interactions that determine the membrane topology of helical peptides (1). Their core consists of alanines, leucine and four histidines arranged to form an amphipathic helix, as well as two lysines at each terminus. Through protonation of its histidines (pKs between 5.4 and 6.0) the alignment of the helices is transmembrane at neutral pH and in-plane at pH <5.5 (1). The LAH4 peptides exhibit membrane poreformation and antimicrobial action at both neutral and at acidic pH including against clinical isolates where the low pH configuration is more active (2). The LAH4 peptides have been found to also exhibit potent DNA and siRNA transfection activities (3). Therefore they can act as a non-viral vector and has indeed been used for the delivery of quantum dots or protein-based vaccines. Furthermore, transduction by adeno-associated viruses or lentiviruses is enhanced by LAH4 (4) or non-peptidic mimetics of this family of peptides (5). Recent and ongoing biophysical, structural and cell biological investigations will be reported which aim to understand these activities at atomic resolution (3, 6-8). (1) B. Bechinger, J.Mol.Biol. 263, 768 (1996). (2) A. J. Mason, et al., J. Biol. Chem. 284, 119 (2009). (3) B. Bechinger, et al. J Pept Sci (2017, in prep.). (4) S. Majdoul, Seye, A.K., Kichler, A., Holic, N., Galy, A., Bechinger, B., Fenard, D., J. Biol. Chem., 291, 2161 (2016) (5) C. Douat, C. et al. Angew. Chem Int. Ed 54, 11133 (2015) (6) C. Aisenbrey, B. Bechinger, Langmuir 30, 10374 (2014). (7) R. Macha´n, P. Jurkiewicz, T. Steinberger, B. Bechinger, M. Hof, Langmuir 30, 6171 (2014). (8) A. Farrotti, G. Bocchinfuso, A. Palleschi, N. Rosato, E. S. Salnikov, N. Voievoda, B. Bechinger, L. Stella, BBA 1848, 581 (2015).
Platform: Micro- and Nanotechnology 127-Plat Improving the Temporal Resolution of Nanopore Recordings Siddharth Shekar1, Chen-Chi Chien2, David Niedzwiecki2, Marija Drndic2, Kenneth Shepard1. 1 Electrical Engineering, Columbia University, New York, NY, USA, 2 Physics and Astronomy, University of Pennsylvania, Philadelphia, PA, USA. Solid-state nanopores are being pursued for a number of applications including, most notably, DNA sequencing. One of the challenges that
Sunday, February 12, 2017
(2) Stranberg E, Zerweck J et al. Biophys J (2013) 104(6):L9-11 (3) Leveritt JM 3rd, Pino-Angeles A and Lazaridis T. Biophys J (2015) 108(10):2424-6 (4) Wiedman G, Fuselier T et al. J Am Chem Soc (2014) 136(12):4724-31 123-Plat Spontaneous Formation of an Ensemble of Structurally Diverse Membrane Channel Architectures from a Single Antimicrobial Peptide Maculatin Yukun Wang1, Charles Chen1, Dan Hu2, Jakob Ulmschneider2, Martin Ulmschneider1. 1 Johns Hopkins University, Baltimore, MD, USA, 2Shanghai Jiaotong University, Shanghai, China. Antimicrobial peptides (AMPs) are an ancient, powerful, and ubiquitous component of the innate immune defence in all domains of life and play a key role in controlling the human microbiome. Many AMPs are known to selectively target and form pores in microbial membranes, killing a wide variety of pathogens at low micromolar concentrations. Fundamental questions remain, however, regarding the molecular mechanisms of membrane targeting, pore formation and function, and the extent to which poration contributes towards antimicrobial activity. Here we report an experimentally guided and validated unbiased long-timescale simulation methodology that yields the complete mechanism of spontaneous pore assembly in the membrane for the amphiphilic pore-forming AMP maculatin at atomic resolution. Rather than a single well defined pore, maculatin was found to form an ensemble of well defined temporarily functional channels that continuously form and dissociate in the membrane. All channels are formed from highly symmetric low-oligomeric assemblies of up to 8 membrane-spanning peptides that mimic integral membrane protein channels in structure. Each channel has a different architecture, functional lifetime, and ion conductivity and overall membrane permeabilization is dominated by higher order oligomers, which form ion channels that also conduct water at high rates. The highest order oligomers were also found to efficiently conduct small dyes. Channel architectures as well as their relative weighting in the ensemble are sensitive to minor mutations as well as variation of lipid tail length. All pores are formed spontaneously by the consecutive addition of individual helices to a transmembrane helix or helix bundle, in contrast to current poration models postulated for AMPs. The diversity of channel architectures formed by a single sequence is remarkable and could explain why no sequence-function relationships in AMP sequences have been discovered to date. Structural ensembles formed by AMP may be the key to preventing bacterial resistance. 124-Plat An Exact Model of Daptomycin Binding to Lipid Bilayers Antje Pokorny, Tala O. Khatib. Chemistry and Biochemistry, Univ. North Carolina Wilmington, Wilmington, NC, USA. Daptomycin is a cyclic lipopeptide of clinical importance in the treatment of multi-drug resistant infections, including those caused by methicillinresistant S. aureus (MRSA) strains. Similar to other antimicrobial peptides, daptomycin binds with preference to the anionic cytoplasmic membranes typically found in prokaryotes. However, in contrast to most linear, alpha-helical peptides, daptomycin binds to lipid bilayers only in the presence of calcium ions and its activity is absolutely calcium-dependent. We measured the interaction of daptomycin with anionic lipid membranes using kinetic binding experiments and equilibrium titrations. The data were analyzed using an exact model describing the interactions of daptomycin with the lipid bilayer that includes solution and membrane-bound states, and the influence of calcium ions on daptomycin-lipid interactions. 125-Plat The Metallopeptides Piscidin 1 and Piscidin 3 Employ Membrane and Nuclease Activity to Eradicate Planktonic, Biofilm, and Persister Cells Myriam L. Cotten1, M. Daben J. Libardo2, Ali Adem Bahar3, Riqiang Fu4, Vitalii I. Silin5, Dacheng Ren3, Mihaela Mihailescu5, Alfredo Angeles-Boza2. 1 Applied Science, College of William and Mary, Williamsburg, VA, USA, 2 Chemistry, University of Connecticut, Storrs, CT, USA, 3Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY, USA, 4National High Magnetic Field Laboratory, Tallahassee, FL, USA, 5Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA. Piscidin 1 (P1) and piscidin 3 (P3) are highly potent host-defense peptides (HDPs) active against drug resistant bacteria, and therefore important for the design of novel anti-infective therapeutics. Both peptides are cationic and interact with the anionic lipid membranes of bacteria. They also translocate across these membranes and colocalize with intracellular DNA. While both peptides have similar three-dimensional structures, P1 is generally more
biologically active than P3 for reasons that are not understood. Here, we compare the disruptive effects of P1 and P3 on lipid bilayers and DNA, and investigate the role of their amino terminal Cu and Ni (ATCUN) binding motif in their mechanism of action. We rely on a combination of complementary methods, including CD- and NMR-monitored titrations, structural solid-state NMR, neutron diffraction with deuterium labeling, surface plasmon resonance, microscopy, biological activity assays, and voltammetry. We find by solid-state NMR that both peptides use an a-helical structure to interact with bacterial cell membrane mimics and isolated DNA. Interestingly, while P1 is more membrane active than P3, the latter is more disruptive to DNA. Through neutron diffraction studies, we demonstrate that P1 inserts more deeply in lipid membranes and induces a major conformational rearrangement of the lipid headgroups, which leads to bilayer defects and significant water penetration into the interstices of the membrane. Remarkably, both peptides bound to Cu2þ act as nucleases that damage isolated DNA within minutes and P3 is significantly more effective than P1. In the context of bacterial cells, we show that copper plays an essential role in planktonic, biofilm, and persister cell eradication. Furthermore, we find that the more membrane-active P1 kills bacteria rapidly while the more DNA-damaging P3 results in slower cell death. Taken together, these results identify host defense metallopeptides with complementary and multi-pronged mechanisms of action as valuable templates to consider for the design of novel strategies against drug resistant bacteria. 126-Plat 20D Years and no End in Sight: Histidine-Rich Designer Peptides with pH-Dependent Membrane Topology and with Multifacet Biomedical Potential Christopher Aisenbrey1, Philippe Bertani1, David Fenard2, Anne Galy3, Elise Glattard1, Martin Gotthardt4, Antoine Kichler5, Nan Liu4, Arnaud Marquette1, Regine S€uss4, Louic Vermeer1, Dennis Wilkins-Juhl1, Justine Wolf1, Burkhard Bechinger1. 1 Chemistry, University of Strasbourg/CNRS, Strasbourg, France, 2Innovation Technologique Lentivirus, Ge´ne´thon, Evry, France, 3Ge´ne´thon/Inserm UMR951, Evry, France, 4Pharmazeutische Technologie und Biopharmazie, University of Freiburg, Freiburg, Germany, 5Laboratoire ‘‘Vecteurs: Synthe`se et Applications The´rapeutiques’’ (V-SAT), CNRS, Strasbourg, France. The synthetic LAH4 peptides were designed to investigate the interactions that determine the membrane topology of helical peptides (1). Their core consists of alanines, leucine and four histidines arranged to form an amphipathic helix, as well as two lysines at each terminus. Through protonation of its histidines (pKs between 5.4 and 6.0) the alignment of the helices is transmembrane at neutral pH and in-plane at pH <5.5 (1). The LAH4 peptides exhibit membrane poreformation and antimicrobial action at both neutral and at acidic pH including against clinical isolates where the low pH configuration is more active (2). The LAH4 peptides have been found to also exhibit potent DNA and siRNA transfection activities (3). Therefore they can act as a non-viral vector and has indeed been used for the delivery of quantum dots or protein-based vaccines. Furthermore, transduction by adeno-associated viruses or lentiviruses is enhanced by LAH4 (4) or non-peptidic mimetics of this family of peptides (5). Recent and ongoing biophysical, structural and cell biological investigations will be reported which aim to understand these activities at atomic resolution (3, 6-8). (1) B. Bechinger, J.Mol.Biol. 263, 768 (1996). (2) A. J. Mason, et al., J. Biol. Chem. 284, 119 (2009). (3) B. Bechinger, et al. J Pept Sci (2017, in prep.). (4) S. Majdoul, Seye, A.K., Kichler, A., Holic, N., Galy, A., Bechinger, B., Fenard, D., J. Biol. Chem., 291, 2161 (2016) (5) C. Douat, C. et al. Angew. Chem Int. Ed 54, 11133 (2015) (6) C. Aisenbrey, B. Bechinger, Langmuir 30, 10374 (2014). (7) R. Macha´n, P. Jurkiewicz, T. Steinberger, B. Bechinger, M. Hof, Langmuir 30, 6171 (2014). (8) A. Farrotti, G. Bocchinfuso, A. Palleschi, N. Rosato, E. S. Salnikov, N. Voievoda, B. Bechinger, L. Stella, BBA 1848, 581 (2015).
Platform: Micro- and Nanotechnology 127-Plat Improving the Temporal Resolution of Nanopore Recordings Siddharth Shekar1, Chen-Chi Chien2, David Niedzwiecki2, Marija Drndic2, Kenneth Shepard1. 1 Electrical Engineering, Columbia University, New York, NY, USA, 2 Physics and Astronomy, University of Pennsylvania, Philadelphia, PA, USA. Solid-state nanopores are being pursued for a number of applications including, most notably, DNA sequencing. One of the challenges that