Gazit E, Boman A, Boman H G, Shai Y
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Biochemistry. 1995 Sep 12;34(36):11479-88. doi: 10.1021/bi00036a021.
Cecropins are positively charged antibacterial polypeptides that were originally isolated from insects. Later on a mammalian homologue, cecropin P1 (CecP), was isolated from pig intestines. While insect cecropins are highly potent against both Gram-negative and Gram-positive bacteria, CecP is as active as insect cecropins against Gram-negative but has reduced activity against Gram-positive bacteria. To gain insight into the mechanism of action of CecP and the molecular basis of its antibacterial specificity, the peptide and its proline incorporated analogue (at the conserved position found in insect cecropins), P22-CecP, were synthesized and labeled on their N-terminal amino-acids with fluorescent probes, without significantly affecting their antibacterial activities. Fluorescence studies indicated that the N-terminal of CecP is located on the surface of phospholipid membranes. Binding experiments revealed that CecP binds acidic phosphatidylserine/phosphatidylcholine (PS/PC) vesicles better than zwitterionic PC vesicles, which correlates with its ability to permeate the former better than the latter. The shape of the binding isotherms suggest that CecP, like insect cecropin, binds phospholipids in a simple, noncooperative manner. However, resonance energy transfer (RET) measurements revealed that, unlike insect cecropins, CecP does not aggregate in the membrane even at relatively high peptide to lipid ratios. The stoichiometry of CecP binding to vesicles suggests that amount of CecP sufficient to form a monolayer causes vesicle permeation. In spite of the incorporation of the conserved proline in P22-CecP, the analogue has reduced antibacterial activity, which correlates with its reduced alpha-helical structure and its lower partitioning and membrane permeating activity with phospholipid vesicles. Taken together, our results support a mechanism in which CecP disrupts the structure of the bacterial membrane by (i) binding of peptide monomers to the acidic surface of the bacterial membrane and (ii) disintegrating the bacterial membrane by disrupting the lipid packing in the bilayers. These results, combined with data reported for other antibacterial polypeptides, suggest that the organization of peptide monomers within phospholipid membranes contributes to Gram-positive/Gram-negative antibacterial specificity.
天蚕素是一类带正电荷的抗菌多肽，最初从昆虫中分离得到。后来，一种哺乳动物同源物——天蚕素P1（CecP），从猪肠道中分离出来。昆虫天蚕素对革兰氏阴性菌和革兰氏阳性菌都具有高效抗菌活性，而CecP对革兰氏阴性菌的活性与昆虫天蚕素相当，但对革兰氏阳性菌的活性有所降低。为了深入了解CecP的作用机制及其抗菌特异性的分子基础，合成了该多肽及其脯氨酸掺入类似物（在昆虫天蚕素的保守位置）P22-CecP，并在其N端氨基酸上用荧光探针标记，且未显著影响它们的抗菌活性。荧光研究表明，CecP的N端位于磷脂膜表面。结合实验表明，CecP与酸性磷脂酰丝氨酸/磷脂酰胆碱（PS/PC）囊泡的结合优于两性离子PC囊泡，这与其穿透前者的能力优于后者相关。结合等温线的形状表明，CecP与昆虫天蚕素一样，以简单、非协同的方式结合磷脂。然而，共振能量转移（RET）测量表明，与昆虫天蚕素不同，即使在相对较高的肽与脂质比例下，CecP也不会在膜中聚集。CecP与囊泡结合的化学计量表明，足以形成单层的CecP量会导致囊泡渗透。尽管P22-CecP中掺入了保守的脯氨酸，但该类似物的抗菌活性降低，这与其α-螺旋结构减少以及与磷脂囊泡的分配和膜渗透活性较低相关。综上所述，我们的结果支持一种机制，即CecP通过（i）肽单体与细菌膜酸性表面的结合以及（ii）通过破坏双层膜中的脂质堆积来瓦解细菌膜，从而破坏细菌膜的结构。这些结果与其他抗菌多肽的报道数据相结合，表明磷脂膜内肽单体的组织有助于革兰氏阳性/革兰氏阴性抗菌特异性。
