Park C B, Yi K S, Matsuzaki K, Kim M S, Kim S C
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Yusong-gu, Kusong-dong, Taejon 305-701, Korea.
Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8245-50. doi: 10.1073/pnas.150518097.
Buforin II is a 21-aa potent antimicrobial peptide that forms, in a hydrophobic medium, an amphipathic structure consisting of an N-terminal random coil region (residues 1-4), an extended helical region (residues 5-10), a hinge (residue 11), and a C-terminal regular alpha-helical region (residues 12-21). To elucidate the structural features of buforin II that are required for its potent antimicrobial activity, we synthesized a series of N- and C-terminally truncated or amino acid-substituted synthetic buforin II analogs and examined their antimicrobial activity and mechanism of action. Deletion of the N-terminal random coil region increased the antibacterial activity approximately 2-fold, but further N-terminal truncation yielded peptide analogs with progressively decreasing activity. Removal of four amino acids from the C-terminal end of buforin II resulted in a complete loss of antimicrobial activity. The substitution of leucine for the proline hinge decreased significantly the antimicrobial activity. Confocal fluorescence microscopic studies showed that buforin II analogs with a proline hinge penetrated the cell membrane without permeabilization and accumulated in the cytoplasm. However, removal of the proline hinge abrogated the ability of the peptide to enter cells, and buforin II analogs without a proline hinge localized on the cell surface, permeabilizing the cell membrane. In addition, the cell-penetrating efficiency of buforin II and its truncated analogs, which depended on the alpha-helical content of the peptides, correlated linearly with their antimicrobial potency. Our results demonstrate clearly that the proline hinge is responsible for the cell-penetrating ability of buforin II, and the cell-penetrating efficiency determines the antimicrobial potency of the peptide.
蟾蜍抗菌肽II是一种由21个氨基酸组成的强效抗菌肽，在疏水介质中形成两亲性结构，该结构由N端无规卷曲区域（第1 - 4位残基）、延伸的螺旋区域（第5 - 10位残基）、一个铰链（第11位残基）和C端规则的α螺旋区域（第12 - 21位残基）组成。为了阐明蟾蜍抗菌肽II发挥强效抗菌活性所需的结构特征，我们合成了一系列N端和C端截短或氨基酸取代的蟾蜍抗菌肽II类似物，并检测了它们的抗菌活性和作用机制。删除N端无规卷曲区域可使抗菌活性提高约2倍，但进一步的N端截短会导致肽类似物的活性逐渐降低。从蟾蜍抗菌肽II的C端去除四个氨基酸会导致抗菌活性完全丧失。用亮氨酸取代脯氨酸铰链会显著降低抗菌活性。共聚焦荧光显微镜研究表明，带有脯氨酸铰链的蟾蜍抗菌肽II类似物可穿透细胞膜而不使其透化，并在细胞质中积累。然而，去除脯氨酸铰链会消除该肽进入细胞的能力，且没有脯氨酸铰链的蟾蜍抗菌肽II类似物定位于细胞表面，使细胞膜透化。此外，蟾蜍抗菌肽II及其截短类似物的细胞穿透效率取决于肽的α螺旋含量，与它们的抗菌效力呈线性相关。我们的结果清楚地表明，脯氨酸铰链负责蟾蜍抗菌肽II的细胞穿透能力，而细胞穿透效率决定了该肽的抗菌效力。
