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A kinked antimicrobial peptide from Bombina maxima. I. Three-dimensional structure determined by NMR in membrane-mimicking environments

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Abstract

Maximin-4 is a 27-residue cationic antimicrobial peptide exhibiting selectivity for bacterial cells. As part of the innate defense system in the Chinese red-belly toad, its mode of action is thought to be ion channel or pore formation and dissipation of the electrochemical gradient across the pathogenic cell membrane. Here we present the high-resolution structure of maximin-4 in two different membrane mimetics, sodium dodecyl sulfate micelles and 50% methanol, as determined by 1H solution NMR spectroscopy. In both environments, the peptide chain adopts a helix–break–helix conformation following a highly disordered N-terminal segment. Despite the similarities in the overall topology of the two structures, major differences are observed in terms of the interactions stabilizing the kink region and the arrangement of the four lysine residues. This has a marked influence on the shape and charge distribution of the molecule and may have implications for the bacterial selectivity of the peptide. The solution NMR results are complemented by CD spectroscopy and solid-state NMR experiments in lipid bilayers, both confirming the predominantly helical conformation of the peptide. As a first step in elucidating the membrane interactions of maximin-4, our study contributes to a better understanding of the mode of action of antimicrobial peptides and the factors governing their selectivity.

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Abbreviations

AMP:

Antimicrobial peptide

MBHA:

4-Methylbenzhydrylamine

DMF:

Dimethylformamide

DCM:

Dichloromethane

DCC:

N,N′-dicyclohexylcarbodiimide

HOBt:

1-Hidroxybenzotriazole

TFA:

Trifluoroacetic acid

EDTA:

Ethylenediamine tetraacetic acid

PIPES:

Piperazine-N-N′-bis(2-ethanesulfonic acid)

SDS:

Sodium dodecyl sulfate

DPC:

Dodecylphosphocholine

DPPC:

Dipalmitoylphosphatidylcholine

DPPG:

Dipalmytoylphosphatidylglycerol

DMPC:

Dimyristoylphosphatidylcholine

DMPG:

Dimyristoylphosphatidylglycerol

MLV:

Multilamellar vesicle

HPLC:

High-pressure liquid chromatography

NMR:

Nuclear magnetic resonance

TOCSY:

Total correlation spectroscopy

NOESY:

Nuclear Overhauser effect spectroscopy

HSQC:

Heteronuclear single quantum correlation

ARIA:

Ambiguous restraints for iterative assignment

RMSD:

Root mean square deviation

MAS:

Magic-angle spinning

REDOR:

Rotational-echo double resonance

CD:

Circular dichroism

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Acknowledgments

The authors thank Dr. Gábor Mező for helpful discussions on peptide synthesis. O.T. thanks Dr. Stephan Grage (Karlsruhe Institute of Technology) for helpful discussions on the manuscript. Z.B. is grateful for the support of the Bolyai János Fellowship program of the Hungarian Academy of Sciences. This work was supported by grants from the Hungarian Research Fund (OTKA) F68326 and the Hungarian GVOP-3.2.1.-2004-04-0210/3.0 project.

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Authors and Affiliations

  1. Institute of Structural Chemistry, Chemical Research Center of the Hungarian Academy of Sciences, 59-67 Pusztaszeri út, 1025, Budapest, Hungary

    Orsolya Toke & Péter Király

  2. Research Group of Peptide Chemistry, Eötvös Loránd University, Hungarian Academy of Sciences, P.O. Box 32, 1518, Budapest 112, Hungary

    Zoltán Bánóczi & Ferenc Hudecz

  3. Karlsruhe Institute of Technology, Institute for Biological Interfaces (IBG-2), Institute of Organic Chemistry, Karlsruhe, Germany

    Ralf Heinzmann, Jochen Bürck & Anne S. Ulrich

  4. Department of Organic Chemistry, Eötvös Loránd University, P.O. Box 32, 1518, Budapest 112, Hungary

    Ferenc Hudecz

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  1. Orsolya Toke
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  2. Zoltán Bánóczi
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  3. Péter Király
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Correspondence to Orsolya Toke.

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Membrane-active peptides: 455th WE-Heraeus-Seminar and AMP 2010 Workshop.

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Toke, O., Bánóczi, Z., Király, P. et al. A kinked antimicrobial peptide from Bombina maxima. I. Three-dimensional structure determined by NMR in membrane-mimicking environments. Eur Biophys J 40, 447–462 (2011). https://doi.org/10.1007/s00249-010-0657-0

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