Abstract
The antimicrobial peptides Ocellatin-LB1, -LB2 and -F1, isolated from frogs, are identical from residue 1 to 22, which correspond to the -LB1 sequence, whereas -LB2 carries an extra N and -F1 additional NKL residues at their C-termini. Despite the similar sequences, previous investigations showed different spectra of activities and biophysical investigations indicated a direct correlation between both membrane-disruptive properties and activities, i.e., ocellatin-F1 > ocellatin-LB1 > ocellatin-LB2. This study presents experimental evidence as well as results from theoretical studies that contribute to a deeper understanding on how these peptides exert their antimicrobial activities and how small differences in the amino acid composition and their secondary structure can be correlated to these activity gaps. Solid-state NMR experiments allied to the simulation of anisotropic NMR parameters allowed the determination of the membrane topologies of these ocellatins. Interestingly, the extra Asn residue at the Ocellatin-LB2 C-terminus results in increased topological flexibility, which is mainly related to wobbling of the helix main axis as noticed by molecular dynamics simulations. Binding kinetics and thermodynamics of the interactions have also been assessed by Surface Plasmon Resonance and Isothermal Titration Calorimetry. Therefore, these investigations allowed to understand in atomic detail the relationships between peptide structure and membrane topology, which are in tune within the series -F1 > > -LB1 ≥ -LB2, as well as how peptide dynamics can affect membrane topology, insertion and binding.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- ACN:
-
Acetonitrile
- AMBER:
-
Assisted Model Building with Energy Refinement
- AMP:
-
Antimicrobial peptides
- CP:
-
Cross Polarization
- DCM:
-
Dichloromethane
- DIC:
-
N,N’-Diisopropylcarbodiimide
- DMF:
-
N,N’-Dimethylformamide
- EASY:
-
Elimination of Artifacts in NMR Spectroscopy
- EDT:
-
1,2-Ethanedithiol
- Fmoc:
-
9-Fluorenylmethoxicarbonyl
- GROMACS:
-
Groningen Machine for Chemical Simulations
- HOBt:
-
1-Hydroxybenzotriazole
- ITC:
-
Isothermal titration calorimetry
- LINCS:
-
Linear Constraint Solver
- LUVs:
-
Large Unilamellar Vesicles
- MD:
-
Molecular Dynamics
- MS:
-
Mass Spectrometry
- NMR:
-
Nuclear Magnetic Resonance
- Oce-LB1:
-
Ocellatin-LB1
- Oce-LB2:
-
Ocellatin-LB2
- Oce-F1:
-
Ocellatin-F1
- NOE:
-
Nuclear Overhauser Effect
- NOESY:
-
Nuclear Overhauser Effect Spectroscopy
- PDB:
-
Protein Data Bank
- POPC:
-
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline
- POPG:
-
1-Palmitoyl-2oleoyl-sn-glycero-3-(phosphor-rac-(1-glycerol))
- RMSD:
-
Root-Mean-Square Deviation
- RU:
-
Reconstructed intensity in relative Units
- SLIPIDS:
-
Stockholm Lipids
- SPR:
-
Surface Plasmon Resonance
- TFA:
-
Trifluoroacetic acid
- TIS:
-
Triisopropylsilane
- ρ:
-
Rotational pitch angle
- σ:
-
Tilt angle
References
Abraham T, Lewis RNAH, Hodges RS, McElhaney RN (2005) Isothermal titration calorimetry studies of the binding of the antimicrobial peptide gramicidin S to phospholipid bilayer membranes. Biochemistry 44:11279–11285. https://doi.org/10.1021/bi050898a
Aisenbrey C, Bechinger B (2004) Tilt and rotational pitch angle of membrane-inserted polypeptides from combined 15N and 2H solid-state NMR spectroscopy. Biochemistry 43:10502–10512. https://doi.org/10.1021/bi049409h
Aisenbrey C, Sizun C, Koch J et al (2006) Structure and dynamics of membrane-associated ICP47, a viral inhibitor of the MHC I antigen-processing machinery. J Biol Chem 281:30365–30372. https://doi.org/10.1074/jbc.M603000200
Aisenbrey C, Bertani P, and Bechinger B (2010) Solid-state NMR of antimicrobial peptides in methods in molecular biology. In: Rinaldi AC, Guiliani A (eds) Antimicrobial Peptides: Methods and Protocols Humana Press, editors vol. 618, pp. 209–233
Aisenbrey C, Michalek M, Salnikov ES, Bechinger B (2019) Solid-state NMR approaches to study protein-structure and protein-lipid interactions. In: Kleinschmitt JH (ed) Methods in Molecular Biology: Lipid-Protein Interactions: Methods and Protocols, Vol 2003, Humana Press, Springer, 974, p 563–598
Bechinger B, Salnikov ES (2012) The membrane interactions of antimicrobial peptides revealed by solid-state NMR spectroscopy. Chem Phys Lipids 165:282–301. https://doi.org/10.1016/j.chemphyslip.2012.01.009
Bechinger B, Sizun C (2003) Alignment and structural analysis of membrane polypeptides by15N and31P solid-state NMR spectroscopy. Concepts Magn Reson Part A Bridg Educ Res 18:130–145. https://doi.org/10.1002/cmr.a.10070
Bechinger B, Resende JM, Aisenbrey C (2011) The structural and topological analysis of membrane-associated polypeptides by oriented solid-state NMR spectroscopy: established concepts and novel developments. Biophys Chem 153:115–125. https://doi.org/10.1016/j.bpc.2010.11.002
Berendsen HJC, van der Spoel D, van Drunen R (1995) GROMACS: a message-passing parallel molecular dynamics implementation. Comput Phys Commun 91:43–56. https://doi.org/10.1016/0010-4655(95)00042-E
Bong DT, Janshoff A, Steinem C, Ghadiri MR (2000) Membrane partitioning of the cleavage peptide in flock house virus. Biophys J 78:839–845. https://doi.org/10.1016/S0006-3495(00)76641-0
Brotman Y, Makovitzki A, Shai Y et al (2009) Synthetic ultrashort cationic lipopeptides induce systemic plant defense responses against bacterial and fungal pathogens. Appl Environ Microbiol 75:5373–5379. https://doi.org/10.1128/AEM.00724-09
Bussi G, Donadio D, Parrinello M (2007) Canonical sampling through velocity rescaling. J Chem Phys 126:14101. https://doi.org/10.1063/1.2408420
Chibowski E, Szcześ A (2016) Zeta potential and surface charge of DPPC and DOPC liposomes in the presence of PLC enzyme. Adsorption 22:755–765. https://doi.org/10.1007/s10450-016-9767-z
Cunha Neto RS, Vigerelli H, Jared C et al (2015) Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus. J Venom Anim Toxins Incl Trop Dis 21:50. https://doi.org/10.1186/s40409-015-0048-1
Czaplewski L, Bax R, Clokie M et al (2016) Alternatives to antibiotics-a pipeline portfolio review. Lancet Infect Dis 16:239–251. https://doi.org/10.1016/S1473-3099(15)00466-1
Darden T, York D, Pedersen L (1993) Particle mesh Ewald: An N·log(N) method for Ewald sums in large systems. J Chem Phys 98:10089–10092. https://doi.org/10.1063/1.464397
Davis JH, Jeffrey KR, Bloom M et al (1976) Quadrupolar echo deuteron magnetic resonance spectroscopy in ordered hydrocarbon chains. Chem Phys Lett 42:390–394. https://doi.org/10.1016/0009-2614(76)80392-2
De Planque MRR, Killian JA (2003) Protein-lipid interactions studied with designed transmembrane peptides: Role of hydrophobic matching and interfacial anchoring (Review). Mol Membr Biol 20:271–284. https://doi.org/10.1080/09687680310001605352
Dourado FS, Leite JRSA, Silva LP et al (2007) Antimicrobial peptide from the skin secretion of the frog Leptodactylus syphax. Toxicon 50:572–580. https://doi.org/10.1016/j.toxicon.2007.04.027
Frasca V (2016) Biophysical characterization of antibodies with isothermal titration calorimetry. J Appl Bioanal 2:90–102. https://doi.org/10.17145/jab.16.013
Gomes KAGG, dos Santos DM, Santos VM et al (2018) NMR structures in different membrane environments of three ocellatin peptides isolated from Leptodactylus labyrinthicus. Peptides 103:72–83. https://doi.org/10.1016/j.peptides.2018.03.016
Granseth E, Von Heijne G, Elofsson A (2005) A study of the membrane-water interface region of membrane proteins. J Mol Biol 346:377–385. https://doi.org/10.1016/j.jmb.2004.11.036
Gusmão KAG, dos Santos DM, Santos VM et al (2017) Ocellatin peptides from the skin secretion of the South American frog Leptodactylus labyrinthicus (Leptodactylidae): characterization, antimicrobial activities and membrane interactions. J Venom Anim Toxins Incl Trop Dis 23:1–14. https://doi.org/10.1186/s40409-017-0094-y
Hediger S, Meier BH, Ernst RR (1995) Adiabatic passage Hartmann-Hahn cross polarization in NMR under magic angle sample spinning. Chem Phys Lett 240:449–456. https://doi.org/10.1016/0009-2614(95)00505-X
Hess B, Bekker H, Berendsen HJC, Fraaije J (1997) LINCS: a linear constraint solver for molecular simulations. J Comput Chem 18:1463–1472
Humphrey W, Dalke A, Schulten K (1996) VMD: Visual molecular dynamics. J Mol Graph 14:33–38. https://doi.org/10.1016/0263-7855(96)00018-5
Jaeger C, Hemmann F (2014) EASY: A simple tool for simultaneously removing background, deadtime and acoustic ringing in quantitative NMR spectroscopy - Part I: basic principle and applications. Solid State Nucl Magn Reson 57–58:22–28. https://doi.org/10.1016/j.ssnmr.2013.11.002
Jämbeck JPM, Lyubartsev AP (2012) Derivation and systematic validation of a refined All-atom force field for phosphatidylcholine lipids. J Phys Chem B 116:3164–3179. https://doi.org/10.1021/jp212503e
Mason AJ, Moussaoui W, Abdelrahman T et al (2009) Structural determinants of antimicrobial and antiplasmodial activity and selectivity in histidine-rich amphipathic cationic peptides. J Biol Chem 284:119–133. https://doi.org/10.1074/jbc.M806201200
Jorgensen WL, Chandrasekhar J, Madura JD et al (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79:926–935. https://doi.org/10.1063/1.445869
Junior EFC, Guimarães CFRC, Franco LL et al (2017) Glycotriazole-peptides derived from the peptide HSP1: synergistic effect of triazole and saccharide rings on the antifungal activity. Amino Acids 49:1389–1400. https://doi.org/10.1007/s00726-017-2441-2
Kang SJ, Park SJ, Mishig-Ochir T, Lee BJ (2014) Antimicrobial peptides: therapeutic potentials. Expert Rev Anti Infect Ther 12:1477–1486. https://doi.org/10.1586/14787210.2014.976613
Killian JA, Von Heijne G (2000) How proteins adapt to a membrane-water interface. Trends Biochem Sci 25:429–434. https://doi.org/10.1016/S0968-0004(00)01626-1
King JD, Rollins-Smith LA, Nielsen PF et al (2005) Characterization of a peptide from skin secretions of male specimens of the frog, Leptodactylus fallax that stimulates aggression in male frogs. Peptides 26:597–601. https://doi.org/10.1016/j.peptides.2004.11.004
Kougentakis CM, Grasso EM, Robinson AC et al (2018) Anomalous properties of lys residues buried in the hydrophobic interior of a protein revealed with 15N-Detect NMR spectroscopy. J Phys Chem Lett 9:383–387. https://doi.org/10.1021/acs.jpclett.7b02668
Li ZL, Wang JJ, Ding HM, Ma YQ (2014) Influence of different membrane environments on the behavior of cholesterol. RSC Adv 4:53090–53096. https://doi.org/10.1039/c4ra08201j
Libério MS, Joanitti GA, Azevedo RB et al (2011) Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells. Amino Acids 40:51–59. https://doi.org/10.1007/s00726-009-0384-y
Lindorff-Larsen K, Piana S, Palmo K et al (2010) Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins Struct Funct Bioinforma 78:1950–1958. https://doi.org/10.1002/prot.22711
López Cascales JJ, Zenak S, García De La Torre J et al (2018) Small cationic peptides: influence of charge on their antimicrobial activity. ACS Omega 3:5390–5398. https://doi.org/10.1021/acsomega.8b00293
Mahlapuu M, Håkansson J, Ringstad L, Björn C (2016) Antimicrobial peptides: an emerging category of therapeutic agents. Front Cell Infect Microbiol 6:1–12. https://doi.org/10.3389/fcimb.2016.00194
Marani MM, Dourado FS, Quelemes PV et al (2015) Characterization and biological activities of ocellatin peptides from the skin secretion of the frog leptodactylus pustulatus. J Nat Prod 78:1495–1504. https://doi.org/10.1021/np500907t
Michalek M, Salnikov ES, Bechinger B (2013) Structure and topology of the huntingtin 1–17 membrane anchor by a combined solution and solid-state NMR approach. Biophys J 105:699–710
Midura-Nowaczek K, Markowska A (2014) Antimicrobial peptides and their analogs: searching for new potential therapeutics. Perspect Med Chem 6:73–80. https://doi.org/10.4137/PMC.S13215
Nosé S (1984) A molecular dynamics method for simulations in the canonical ensemble. Mol Phys 52:255–268. https://doi.org/10.1080/00268978400101201
Parrinello M, Rahman A (1981) Polymorphic transitions in single crystals: a new molecular dynamics method. J Appl Phys 52:7182–7190. https://doi.org/10.1063/1.328693
Powers JPS, Hancock REW (2003) The relationship between peptide structure and antibacterial activity. Peptides 24:1681–1691. https://doi.org/10.1016/j.peptides.2003.08.023
Rance M, Byrd RA (1983) Obtaining high-fidelity spin-12 powder spectra in anisotropic media: Phase-cycled Hahn echo spectroscopy. J Magn Reson 52:221–240. https://doi.org/10.1016/0022-2364(83)90190-7
Resende JM, Moraes CM, Munhoz VHO et al (2009) Membrane structure and conformational changes of the antibiotic heterodimeric peptide distinctin by solid-state NMR spectroscopy. Proc Natl Acad Sci USA 106:16639–16644. https://doi.org/10.1073/pnas.0905069106
Resende JM, Verly RM, Aisenbrey C et al (2014) Membrane interactions of phylloseptin-1, -2, and -3 peptides by oriented solid-state NMR spectroscopy. Biophys J 107:901–911. https://doi.org/10.1016/j.bpj.2014.07.014
Salnikov E, Bertani P, Raap J, Bechinger B (2009) Analysis of the amide15N chemical shift tensor of the Cαtetrasubstituted constituent of membrane-active peptaibols, the α-aminoisobutyric acid residue, compared to those of di- and tri-substituted proteinogenic amino acid residues. J Biomol NMR 45:373–387. https://doi.org/10.1007/s10858-009-9380-5
Satoh K (1995) Determination of binding constants of Ca2+, Na+, and Cl− ions to liposomal membranes of dipalmitoylphosphatidylcholine at gel phase by particle electrophoresis. Biochim Biophys Acta - Biomembr 1239:239–248. https://doi.org/10.1016/0005-2736(95)00154-U
Schmidtchen A, Pasupuleti M, Malmsten M (2014) Effect of hydrophobic modifications in antimicrobial peptides. Adv Colloid Interface Sci 205:265–274. https://doi.org/10.1016/j.cis.2013.06.009
Seelig J (1977) Deuterium magnetic resonance: theory and application to lipid membranes. Q Rev Biophys 10:353–418. https://doi.org/10.1017/S0033583500002948
Sengupta D, Smith JC, Ullmann GM (2008) Partitioning of amino-acid analogues in a five-slab membrane model. Biochim Biophys Acta - Biomembr 1778:2234–2243. https://doi.org/10.1016/j.bbamem.2008.06.014
Sierra JM, Fusté E, Rabanal F et al (2017) An overview of antimicrobial peptides and the latest advances in their development. Expert Opin Biol Ther 17:663–676. https://doi.org/10.1080/14712598.2017.1315402
Sousa JC, Berto RF, Gois EA et al (2009) Leptoglycin: a new Glycine/Leucine-rich antimicrobial peptide isolated from the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae). Toxicon 54:23–32. https://doi.org/10.1016/j.toxicon.2009.03.011
Strandberg E, Killian JA (2003) Snorkeling of lysine side chains in transmembrane helices: how easy can it get? FEBS Lett 544:69–73. https://doi.org/10.1016/S0014-5793(03)00475-7
Tadros TF, Vladisavljević GT, Kobayashi I et al (2013) Emulsion formation, stability, and rheology. Emuls Form Stab 16:1–75
Ulmschneider JP, Smith JC, Ulmschneider MB et al (2012) Reorientation and dimerization of the membrane-bound antimicrobial peptide PGLa from microsecond all-atom MD simulations. Biophys J 103:472–482. https://doi.org/10.1016/j.bpj.2012.06.040
Verly RM, De Moraes CM, Resende JM et al (2009) Structure and membrane interactions of the antibiotic peptide dermadistinctin K by multidimensional solution and oriented 15N and 31P solid-state NMR spectroscopy. Biophys J 96:2194–2203. https://doi.org/10.1016/j.bpj.2008.11.063
Verly RM, Resende JM, Junior EFC et al (2017) Structure and membrane interactions of the homodimeric antibiotic peptide homotarsinin. Sci Rep 7:1–13. https://doi.org/10.1038/srep40854
Verwey EJW, Overbeek JT, Nes K (1948) Theory of the stability of lyophobic colloids: the interaction of sol particles having an electric double layer. Elsiever Publishing Company
Violette A, Fournel S, Lamour K et al (2006) Mimicking helical antibacterial peptides with nonpeptidic folding oligomers. Chem Biol 13:531–538. https://doi.org/10.1016/j.chembiol.2006.03.009
White SH, Wimley WC (1998) Hydrophobic interactions of peptides with membrane interfaces. Biochim Biophys Acta - Rev Biomembr 1376:339–352. https://doi.org/10.1016/S0304-4157(98)00021-5
Wieprecht T, Beyermann M, Seelig J (1999) Binding of antibacterial magainin peptides to electrically neutral membranes: thermodynamics and structure. Biochemistry 38:10377–10387. https://doi.org/10.1021/bi990913+
Wieprecht T, Seelig JBT-CT in M (2002) Isothermal titration calorimetry for studying interactions between peptides and lipid membranes. In: peptide-Lipid Interactions. Academic Press, pp 31–56
Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395
Acknowledgements
JML and JMR acknowledge grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). JCLO acknowledges grants from CNPq and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). We thank CNPq, FAPEMIG, CAPES and Rede Mineira de Química (RQ-MG) for financial support. BB acknowledges the financial contributions of the Agence Nationale de la Recherche, the University of Strasbourg, the CNRS, the Région Grand-Est and the RTRA International Center of Frontier Research in Chemistry. We also thank the NMR facilities at the University of Strasbourg and at UFMG (LAREMAR), as well as the mass spectrometry facility at UFMG (CELAM).
Funding
Part of this work was supported by Agence Nationale de la Recherche (projects MemPepSyn 14-CE34-0001-01, Biosupramol 17-CE18-0033-3, Naturalarsenal 19-AMRB-0004-02 and the LabEx Chemistry of Complex Systems 10-LABX-0026_CSC).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
None.
Additional information
Communicated by T. Langer.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Muñoz-López, J., Oliveira, J.C.L., Michel, D.A.G.R. et al. Membrane interactions of Ocellatins. Where do antimicrobial gaps stem from?. Amino Acids 53, 1241–1256 (2021). https://doi.org/10.1007/s00726-021-03029-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00726-021-03029-0