Sequence context induced antimicrobial activity: insight into lipopolysaccharide permeabilization
Sequence context induced antimicrobial activity: insight into lipopolysaccharide permeabilization
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DOI：
10.1039/c4mb00111g
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发表时间：
2014-01-01
影响因子：
--
通讯作者：
Bhunia, Anirban
中科院分区：
文献类型：
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作者：
Ghosh, Anirban;Datta, Aritreyee;Bhunia, Anirban
乳铁蛋白（Lactoferrampin，WR 17，Trp 268-Arg 284）是一种抗菌肽，具有显著的抗菌和杀念珠菌活性。然而，没有先前的研究解释WR 17如何透化革兰氏阴性菌的外膜并中和内毒素。在这项研究中，我们使用了一系列的测定，如抗微生物活性，钙黄绿素渗漏，NPN染料摄取和内毒素中和测定，以表明WR 17的序列背景调节其多方面的活性。我们测定了WR 17在LPS中的高分辨率NMR结构，发现N-ter区域形成螺旋（Trp 1-Phe 11）并以45度角定向进入脂多糖（LPS）胶束，而C-ter区域（Lys 13-Arg 17）保持为柔性延伸的无规卷曲。我们还通过计算机分子模型模拟验证了这一结果。等温滴定量热法表明，WR 17及其类似物与LPS的相互作用主要是吸热的性质。利用荧光各向异性和红边激发位移分析等技术，我们揭示了WR 17在LPS中对Trp 1的运动限制。WR 17的Trp 1的吲哚环与LPS的极性头基之间的距离约为7埃，如从插入深度测定获得的。此外，MD模拟表明，在C-末端的K（13）xK（15）xR（17）基序的帮助下实现肽在LPS中的掺入。这种新的锚定“K13 NKSR 17”基序目前正用于我们正在进行的研究，以设计新的抗内毒素分子。
Lactoferrampin (WR17, Trp 268-Arg 284), an antimicrobial peptide, is known to have significant antibacterial and candidacidal activities. However, there are no previous studies explaining how WR17 permeabilizes the outer membrane of Gram negative bacteria and neutralizes endotoxins. In this study we used a series of assays like antimicrobial activity, calcein leakage, NPN dye uptake and endotoxin neutralization assay to show that the sequence context of WR17 modulates its multi-faceted activities. We determined the high resolution NMR structure of WR17 in LPS and found that the N-ter region forms a helix (Trp1-Phe11) and orients itself at an angle of 45 degrees into the lipopolysaccharide (LPS) micelle, whereas the C-ter region (Lys13-Arg17) remains as a flexible extended random coil. We also verified this result through in silico molecular modeling simulation. Isothermal titration calorimetry showed that the interaction of WR17 and its analogues with LPS was primarily endothermic in nature. Using several fluorescence techniques such as anisotropy and red edge excitation shift assay we revealed motional restriction for Trp1 of WR17 in LPS. The distance between the indole ring of Trp1 of WR17 and the polar head group of LPS is around 7 angstrom, as obtained from the depth of insertion assay. Additionally, MD simulation demonstrated that the incorporation of the peptide in LPS is achieved with the help of the K(13)xK(15)xR(17) motif at the C-terminus. This novel anchoring "K13NKSR17" motif is currently being utilized in our ongoing research to design novel anti-endotoxic molecules.