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. 2013 Sep 9;8(9):e72718.
doi: 10.1371/journal.pone.0072718. eCollection 2013.

NMR Structure of temporin-1 Ta in Lipopolysaccharide Micelles: Mechanistic Insight Into Inactivation by Outer Membrane

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Free PMC article

NMR Structure of temporin-1 Ta in Lipopolysaccharide Micelles: Mechanistic Insight Into Inactivation by Outer Membrane

Rathi Saravanan et al. PLoS One. .
Free PMC article

Abstract

Background: Antimicrobial peptides (AMPs) play important roles in the innate defense mechanism. The broad spectrum of activity of AMPs requires an efficient permeabilization of the bacterial outer and inner membranes. The outer leaflet of the outer membrane of Gram negative bacteria is made of a specialized lipid called lipopolysaccharide (LPS). The LPS layer is an efficient permeability barrier against anti-bacterial agents including AMPs. As a mode of protection, LPS can induce self associations of AMPs rendering them inactive. Temporins are a group of short-sized AMPs isolated from frog skin, and many of them are inactive against Gram negative bacteria as a result of their self-association in the LPS-outer membrane.

Principal findings: Using NMR spectroscopy, we have determined atomic resolution structure and characterized localization of temporin-1Ta or TA (FLPLIGRVLSGIL-amide) in LPS micelles. In LPS micelles, TA adopts helical conformation for residues L4-I12, while residues F1-L3 are found to be in extended conformations. The aromatic sidechain of residue F1 is involved in extensive packing interactions with the sidechains of residues P3, L4 and I5. Interestingly, a number of long-range NOE contacts have been detected between the N-terminal residues F1, P3 with the C-terminal residues S10, I12, L13 of TA in LPS micelles. Saturation transfer difference (STD) NMR studies demonstrate close proximity of residues including F1, L2, P3, R7, S10 and L13 with the LPS micelles. Notably, the LPS bound structure of TA shows differences with the structures of TA determined in DPC and SDS detergent micelles.

Significance: We propose that TA, in LPS lipids, forms helical oligomeric structures employing N- and C-termini residues. Such oligomeric structures may not be translocated across the outer membrane; resulting in the inactivation of the AMP. Importantly, the results of our studies will be useful for the development of antimicrobial agents with a broader spectrum of activity.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. tr-NOESY spectra of TA in LPS micelles.
(panel A) A section of the two-dimensional 1H-1H NOESY spectrum of TA, in aqueous solution containing LPS micelles, showing NOE correlation among backbone amide proton resonances. (panel B) A section of the two-dimensional 1H-1H NOESY spectrum of TA, in aqueous solution containing LPS micelles, showing NOE correlation among backbone amide proton resonances (along ω2 dimension) with CαH proton resonances (along ω1 dimension).
Figure 2
Figure 2. NOEs of residue F1 in LPS.
A section of 2-D NOESY spectrum of TA, in aqueous solution containing LPS micelles, showing NOE connectivites from aromatic ring protons of residue F1 (along ω2 dimension) with the upfield shifted aliphatic proton resonances (along ω1 dimension). The long-range NOEs are indicated in red color.
Figure 3
Figure 3. Summary of NOE contacts of TA in LPS micelles.
Bar diagram summarizing type (intra, sequential, medium-range) and number of NOE contacts observed for each amino acid of TA while bound to LPS micelles.
Figure 4
Figure 4. Three-dimensional structure of TA in LPS micelles.
(panel A) Superposition of backbone atoms (N, Cα, C′) of twenty lowest energy conformers of TA for residues 1–13. (panel B) A representative conformation of LPS-bound TA showing sidechain orientation. (panel C) A space-fill representation of plausible packing interactions among the N-terminal residues, F1, L2, P3 and L4, of TA in LPS micelles. Figures were generated by INSIGHT II.
Figure 5
Figure 5. Angular order parameter (S) of TA structure in LPS.
A bar diagram showing angular order parameter of backbone dihedral angles, Φ and Ψ, obtained from conformational ensemble of TA in LPS micelles.
Figure 6
Figure 6. Electrostatic surface potential of TA in LPS micelles.
Surface charge distribution of the helical structure of TA. Surfaces in red, blue and white represent, respectively, negatively charged, positively charged and neutral residues. The figure was generated by PyMOL.
Figure 7
Figure 7. Mode of oligomerization of TA in LPS micelles.
A model of the head-tail dimer of the helical structure of TA (panel A) and the electrostatic surface of the dimeric structure (panel B).
Figure 8
Figure 8. Localization of TA in LPS micelles by STD NMR.
The off-resonance or reference TOCSY spectrum (panel A) and the STD-TOCSY spectrum (panel B) of TA in LPS showing correlations among aliphatic proton resonances. Through bond connectivites detected in STD-TOCSY spectra for amino acids residues of TA are shown by broken lines. STD-TOCSY spectra were acquired in D2O using a spin-lock MLEV17 sequence with a mixing time of 80 ms.

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Grant support

This work is supported by a grant from Ministry of Education (MOE), RG11/12, Singapore. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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