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Peptaibols as membrane-active antibiotics: Solid-state 19F-NMR structure-function analysis using tailor-made CF3-labeled amino acids

Subject Area Biological and Biomimetic Chemistry
Term from 2012 to 2016
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 208935089
 
Final Report Year 2017

Final Report Abstract

Fungi such as Trichoderma are producers of pharmaceutically attractive peptaibols that are endowed with improved biostability due to a high content of unconventional amino acids such as aminoisobutyric acid (Aib). Peptaibols are considered as promising antimicrobial drug candidates in view of their interference with cellular membranes. Knowledge of their lipid interactions and membrane-bound structures is needed for understanding their activity and should be in principle accessible by solid-state NMR. However, these short sequences are rich in Aib and Pro, and they have unknown or non-canonical conformations. Therefore, new strategies are required to maximize the structural information from a minimum number of labeled sites. We thus combined L-trifluoromethyl-bicyclopentyl-glycine, (R)- and (S)-trifluoromethyl-alanine, and 15N-backbone labels, each probing a different direction in the molecule, in order to elucidate the conformation and membrane alignment of harzianin HK- VI (HZ: Ac-UNIIUPLLUPL-ol; U=Aib). For this short sequence of 11 amino acids, a total of 12 orientational constraints were obtained using solid-state 19F- and 15N-NMR. Data analysis revealed a β-bend ribbon spiral (a subtype of the 310 helix) lying parallel to the membrane surface in an S-state. The molecule flipped (but retained its unusual structure) into an inserted I-state in bilayers with a high positive spontaneous lipid curvature. Despite its hydrophobic character and its short length, HZ thus exhibits an alignment behavior that is typically observed for longer and distinctly amphipathic sequences, like α-helical antimicrobial pore-forming peptides. Interestingly, in biological tests we found that this peptaibol did not display any significant antibacterial activity against bacteria, but rather showed inhibitory effects against some plant pathogenic fungi. These results suggest that harzianin HK VI may play a role in the antagonism of the Trichoderma species by having a more specific activity against fungal cell membranes. To support the solid-state NMR analysis in supported bilayers, we also used conventional liquid-state NMR to determine the conformation of HZ in membrane-mimicking environments, i.e. in TFE solution, DPC and SDS micelles. The resulting structures were fully consistent with a β-bend ribbon spiral and could be used as models to assess the solid-state NMR data in the membrane-bound state. We also performed unrestricted MD simulations of a HZ molecule interacting with a DMPC bilayer, and free energies were estimated from HREX simulations. Two types of orientation were identified, similar to the findings with solid-state NMR: an inserted I-state, and a surface-bound S-state. Interestingly, in the latter the HZ molecule is located almost completely within the hydrophobic region of the bilayer, in contrast to the behavior of cationic amphiphilic antimicrobial peptides which usually float in the hydrophobic/hydrophilic interface. Most notably, the MD simulations showed that the presence of HZ in the I-state induces a local thinning of the bilayer, which is an important aspect of it being able to span the membrane. To support the solid-state NMR analysis of peptaibols, we have also characterized the suitability of branched-chain diphytanoyl lipids as a highly stable membrane matrix. They were used not only to resolve the unusual structure of the short HZ, but also of the C-terminus of alamethicin in oriented DMPC bilayers, which revealed in addition a lipid phase-dependent re-alignment of this long and well-known peptaibol.

Publications

  • (2017) Diphytanoyl lipids as model systems for studying membrane-active peptides. Biochimica et biophysica acta. Biomembranes 1859 (10) 1828–1837
    Kara, Sezgin; Afonin, Sergii; Babii, Oleg; Tkachenko, Anton N.; Komarov, Igor V.; Ulrich, Anne S.
    (See online at https://doi.org/10.1016/j.bbamem.2017.06.003)
  • (2017) Structural Behavior of the Peptaibol Harzianin HK VI in a DMPC Bilayer: Insights from MD Simulations. Biophysical journal 112 (12) 2602–2614
    Putzu, Marina; Kara, Sezgin; Afonin, Sergii; Grage, Stephan L.; Bordessa, Andrea; Chaume, Grégory; Brigaud, Thierry; Ulrich, Anne S.; Kubař, Tomáš
    (See online at https://doi.org/10.1016/j.bpj.2017.05.019)
  • (2018) Orthogonal 19 F-Labeling for Solid-State NMR Spectroscopy Reveals the Conformation and Orientation of Short Peptaibols in Membranes. Chemistry (Weinheim an der Bergstrasse, Germany) 24 (17) 4328–4335
    Grage, Stephan L.; Kara, Sezgin; Bordessa, Andrea; Doan, Véronique; Rizzolo, Fabio; Putzu, Marina; Kubař, Tomáš; Papini, Anna Maria; Chaume, Grégory; Brigaud, Thierry; Afonin, Sergii; Ulrich, Anne S.
    (See online at https://doi.org/10.1002/chem.201704307)
 
 

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