Lee Sung-Ah, Kim Yu Kyoung, Lim Shin Saeng, Zhu Wan Long, Ko Hyunsook, Shin Song Yub, Hahm Kyung-Soo, Kim Yangmee
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, IBST, Konkuk University, Kwangjin-gu, Seoul 143-701, Korea.
Biochemistry. 2007 Mar 27;46(12):3653-63. doi: 10.1021/bi062233u. Epub 2007 Mar 1.
Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic alpha-helical structure that was isolated from the mast cells of hybrid striped bass [Silphaduang, U., and Noga, E. J. (2001) Nature 414, 268-269]. Pis-1 is not selective for bacterial versus mammalian cells. In the present study, to develop novel antibiotic peptides with selectivity for bacterial cells, we examined the effect of substituting two glycine residues, Gly8 and Gly13, with Ala or Pro on this peptide's structure and biological activities. The bacterial cell selectivity of the peptides decreased in the following order: Gly-->Pro analogues > Gly-->Pro/Ala analogues > Pis-1 > Gly-->Ala analogues. The antimicrobial and hemolytic activities and abilities to permeabilize the model phospholipid membranes were higher for Pis-1 with Gly or Pro at position 8 than for its counterparts with either Gly or Pro at position 13. We determined the tertiary structure of Pis-1 and its analogues in the presence of SDS micelles by NMR spectroscopy. We found that Pis-1 has an alpha-helical structure from Phe2 to Thr21. Also, Pis-1 AA (Gly8, Gly13-->Ala8, Ala13) with higher antibacterial and hemolytic activity than Pis-1 has a stable alpha-helical structure from Phe2 to Thr21. Pis-1 PG (Gly-->Pro8) with bacterial cell selectivity has a hinge structure at Pro8, which provides flexibility in piscidin, followed by a three-turn helix from Val10 to Gly22 in the C-terminal region. Taken together, our results demonstrate that the conformational flexibility provided by introduction of a Pro at position 8, coupled with the primary anchoring of phenylalanines and histidines in the N-terminus to the cell membrane and the optimal length of the C-terminal amphipathic alpha-helix, are the critical factors that confer antibacterial activity and bacterial cell selectivity to Pis-1 PG. Pis-1 PG may be a good candidate for the development of a new drug with potent antibacterial activity but without cytotoxicity.
杀鱼毒素1（Pis-1）是一种具有阳离子α-螺旋结构的新型细胞毒性肽，它是从杂交条纹鲈的肥大细胞中分离出来的[Silphaduang, U., and Noga, E. J. (2001) Nature 414, 268 - 269]。Pis-1对细菌细胞和哺乳动物细胞没有选择性。在本研究中，为了开发对细菌细胞具有选择性的新型抗生素肽，我们研究了用丙氨酸或脯氨酸取代两个甘氨酸残基（Gly8和Gly13）对该肽的结构和生物学活性的影响。肽对细菌细胞的选择性按以下顺序降低：甘氨酸→脯氨酸类似物＞甘氨酸→脯氨酸/丙氨酸类似物＞Pis-1＞甘氨酸→丙氨酸类似物。在第8位带有甘氨酸或脯氨酸的Pis-1的抗菌、溶血活性以及使模型磷脂膜通透的能力，高于在第13位带有甘氨酸或脯氨酸的对应物。我们通过核磁共振光谱法确定了在十二烷基硫酸钠胶束存在下Pis-1及其类似物的三级结构。我们发现Pis-1从Phe2到Thr21具有α-螺旋结构。此外，与Pis-1相比具有更高抗菌和溶血活性的Pis-1 AA（Gly8, Gly13→Ala8, Ala13）从Phe2到Thr21具有稳定的α-螺旋结构。具有细菌细胞选择性的Pis-1 PG（Gly→Pro8）在Pro8处有一个铰链结构，这为杀鱼毒素提供了灵活性，随后在C端区域从Val10到Gly22有一个三圈螺旋。综上所述，我们的结果表明，在第8位引入脯氨酸所提供的构象灵活性，加上N端的苯丙氨酸和组氨酸与细胞膜的主要锚定作用以及C端两亲性α-螺旋的最佳长度，是赋予Pis-1 PG抗菌活性和细菌细胞选择性的关键因素。Pis-1 PG可能是开发一种具有强效抗菌活性但无细胞毒性的新药的良好候选物。
