School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
PLoS One. 2013 Sep 9;8(9):e72718. doi: 10.1371/journal.pone.0072718. eCollection 2013.
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.
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.
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.
抗菌肽（AMPs）在先天防御机制中发挥重要作用。AMPs 的广谱活性需要有效地透化细菌的外膜和内膜。革兰氏阴性细菌外膜的外叶由一种称为脂多糖（LPS）的特殊脂质组成。LPS 层是对抗包括 AMP 在内的抗菌剂的有效渗透屏障。作为一种保护机制，LPS 可以诱导 AMP 发生自我缔合，从而使它们失去活性。蛙皮素是从蛙皮中分离出的一组短肽 AMP，由于它们在 LPS-外膜中的自我缔合，许多蛙皮素对革兰氏阴性菌没有活性。
使用 NMR 光谱学，我们确定了在 LPS 胶束中 temporin-1Ta 或 TA（FLPLIGRVLSGIL-amide）的原子分辨率结构，并对其定位进行了表征。在 LPS 胶束中，TA 的残基 L4-I12 呈螺旋构象，而残基 F1-L3 则呈伸展构象。残基 F1 的芳基侧链与残基 P3、L4 和 I5 的侧链发生广泛的堆积相互作用。有趣的是，在 LPS 胶束中检测到 TA 的 N 端残基 F1、P3 与 C 端残基 S10、I12、L13 之间存在大量的长程 NOE 接触。饱和转移差异（STD）NMR 研究表明，包括 F1、L2、P3、R7、S10 和 L13 在内的残基与 LPS 胶束接近。值得注意的是，TA 与 LPS 结合的结构与在 DPC 和 SDS 去污剂胶束中确定的 TA 结构存在差异。
我们提出，TA 在 LPS 脂质中形成螺旋寡聚体结构，利用 N-和 C-末端残基。这种寡聚体结构可能不会穿过外膜；从而使 AMP 失活。重要的是，我们的研究结果将有助于开发具有更广泛活性谱的抗菌剂。
