Ghosh Anirban, Datta Aritreyee, Jana Jagannath, Kar Rajiv Kumar, Chatterjee Chiradip, Chatterjee Subhrangsu, Bhunia Anirban
Biomolecular NMR and Drug Design Laboratory, Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India.
Mol Biosyst. 2014 Jun;10(6):1596-612. doi: 10.1039/c4mb00111g. Epub 2014 Apr 9.
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° 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 Å, 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 "K(13)NKSR(17)" motif is currently being utilized in our ongoing research to design novel anti-endotoxic molecules.
乳铁杀菌肽（WR17，色氨酸268 - 精氨酸284）是一种抗菌肽，已知具有显著的抗菌和抗念珠菌活性。然而，以前没有研究解释WR17如何使革兰氏阴性菌的外膜通透性增加并中和内毒素。在本研究中，我们使用了一系列测定方法，如抗菌活性、钙黄绿素泄漏、NPN染料摄取和内毒素中和测定，以表明WR17的序列背景调节其多方面的活性。我们确定了WR17在脂多糖中的高分辨率核磁共振结构，发现N端区域形成一个螺旋（色氨酸1 - 苯丙氨酸11），并以45°角定向进入脂多糖（LPS）胶束，而C端区域（赖氨酸13 - 精氨酸17）保持为灵活的延伸无规卷曲。我们还通过计算机分子模拟验证了这一结果。等温滴定量热法表明，WR17及其类似物与LPS的相互作用本质上主要是吸热的。使用几种荧光技术，如各向异性和红边激发位移测定，我们揭示了WR17的色氨酸1在LPS中的运动受限。从插入深度测定获得，WR17的色氨酸1的吲哚环与LPS的极性头部基团之间的距离约为7 Å。此外，分子动力学模拟表明，肽在LPS中的掺入是借助C端的K(13)xK(15)xR(17)基序实现的。这种新型的锚定“K(13)NKSR(17)”基序目前正在我们正在进行的研究中用于设计新型抗内毒素分子。
